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1.
Gene ; 743: 144624, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32224274

RESUMO

The giant ciliate Stentor coeruleus (S. coeruleus) is a suitable model organism for studying morphogenesis and regeneration at the single-cell level. It contains a prominent structure on the anterior end of the cell, known as the oral apparatus (OA). OA can be induced to shed by urea treatment and then new OA regenerates via a series of defined morphological events and the cell resumes normal feeding activity. We identified OA constituents in S. coeruleus by mass spectrometry. A total of 882 OA-associated proteins were identified; the homologs of 181 of these are known OA constituents in other organisms. The expression pattern of OA-associated genes during regeneration was investigated using single-cell transcriptome sequencing. The expression of most OA-associated genes was high during regeneration, indicating their stable expression after OA shedding. We also identified OA-associated differentially expressed genes that may be involved in regulating OA reconstruction. In summary, this study gives preliminary insight into the molecular basis of OA in S. coeruleus.


Assuntos
Cilióforos/fisiologia , Genes de Protozoários/genética , Proteínas de Protozoários/metabolismo , Regeneração , Espectrometria de Massas , Proteômica , Proteínas de Protozoários/genética , Análise de Sequência de RNA , Análise de Célula Única
2.
PLoS Genet ; 16(2): e1008390, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32084124

RESUMO

Base J, ß-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.


Assuntos
DNA de Cinetoplasto/metabolismo , Proteínas de Protozoários/metabolismo , RNA Polimerase II/metabolismo , Terminação da Transcrição Genética , Trypanosoma brucei brucei/fisiologia , Animais , DNA de Cinetoplasto/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes de Protozoários , Glucosídeos/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Leishmania/fisiologia , Mutação , Proteínas de Protozoários/genética , Interferência de RNA , RNA Polimerase II/genética , Timina/metabolismo , Uracila/análogos & derivados , Uracila/metabolismo
3.
Parasitol Res ; 119(3): 1101-1108, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32006227

RESUMO

Limited data are available on infection rates and genetic identity of Enterocytozoon bieneusi and Giardia duodenalis in horses and donkeys. In this study, 865 fecal specimens were collected from donkeys (n = 540) and horses (n = 325) in three provinces and autonomous regions in northern China during 2015-2019. Enterocytozoon bieneusi was detected and genotyped by PCR and sequence analyses of the ribosomal internal transcribed spacer (ITS) and G. duodenalis was detected and genotyped by PCR and sequence analyses of the ß-giardin, glutamate dehydrogenase, and triosephosphate isomerase genes. The overall infection rates of E. bieneusi and G. duodenalis were 21.9% (118/540) and 11.5% (62/540) in donkeys, and 7.4% (24/325) and 2.8% (9/325) in horses, respectively. These differences in infection rates of E. bieneusi and G. duodenalis between donkeys and horses were significant (χ2 = 30.9, df = 1, P < 0.0001; χ2 = 20.4, df = 1, P < 0.0001, respectively). By age, the 28.9% infection rate of E. bieneusi in donkeys under 6 months was significantly higher than that in animals over 6 months (6.0%; χ2 = 35.2, df = 1, P < 0.0001). In contrast, donkeys of 6-12 months had higher infection rate (35.9%) of G. duodenalis than donkeys under 6 months (9.9%; χ2 = 22.1, df = 1, P < 0.0001) and over 12 months (8.7%; χ2 = 17.3, df = 1, P < 0.0001). In horses, animals of > 12 months had significantly higher infection rate (31.1%) of E. bieneusi than horses under 6 months (3.4%; χ2 = 29.4, df = 1, P < 0.0001) and 6-12 months (3.8%; χ2 = 26.1, df = 1, P < 0.0001). Twenty genotypes of E. bieneusi were detected, including six known ones and 14 new genotypes. Among them, nine genotypes in 45% E. bieneusi-positive specimens belonged to the zoonotic group 1. Similarly, three G. duodenalis assemblages were detected, including A (in 2 horses and 30 donkeys), B (in 6 horses and 29 donkeys), and E (in 1 horse); three donkeys had coinfections of assemblages A and B. The assemblage A isolates identified all belong to the sub-assemblage AI. These results indicate that unlike in other farm animals, there is a common occurrence of zoonotic E. bieneusi and G. duodenalis genotypes in horses and donkeys.


Assuntos
Enterocytozoon/fisiologia , Equidae/parasitologia , Giardia lamblia/fisiologia , Giardíase/veterinária , Cavalos/parasitologia , Microsporidiose/veterinária , Animais , Animais Domésticos/parasitologia , China/epidemiologia , Enterocytozoon/classificação , Enterocytozoon/genética , Fezes/parasitologia , Genes de Protozoários/genética , Genótipo , Giardia lamblia/classificação , Giardia lamblia/genética , Giardíase/epidemiologia , Giardíase/parasitologia , Especificidade de Hospedeiro , Microsporidiose/epidemiologia , Microsporidiose/parasitologia , Filogenia , Prevalência , Zoonoses/transmissão
4.
PLoS Negl Trop Dis ; 14(1): e0007770, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32004318

RESUMO

BACKGROUND: Genetic exchange in Trypanosoma cruzi is controversial not only in relation to its frequency, but also to its mechanism. Parasexual genetic exchange has been proposed based on laboratory hybrids, but population genomics strongly suggests meiosis in T. cruzi. In addition, mitochondrial introgression has been reported several times in natural isolates although its mechanism is not fully understood yet. Moreover, hybrid T. cruzi DTUs (TcV and TcVI) have inherited at least part of the kinetoplastic DNA (kDNA = mitochondrial DNA) from both parents. METHODOLOGY/PRINCIPAL FINDINGS: In order to address such topics, we sequenced and analyzed fourteen nuclear DNA fragments and three kDNA maxicircle genes in three TcI stocks which are natural clones potentially involved in events of genetic exchange. We also deep-sequenced (a total of 6,146,686 paired-end reads) the minicircle hypervariable region (mHVR) of the kDNA in such three strains. In addition, we analyzed the DNA content by flow cytometry to address cell ploidy. We observed that most polymorphic sites in nuclear loci showed a hybrid pattern in one cloned strain and the other two cloned strains were compatible as parental strains (or nearly related to the true parents). The three clones had almost the same ploidy and the DNA content was similar to the reference strain Sylvio (a nearly diploid strain). Despite maxicircle genes evolve faster than nuclear housekeeping ones, we detected no polymorphisms in the sequence of three maxicircle genes showing mito-nuclear discordance. Lastly, the hybrid stock shared 66% of its mHVR clusters with one putative parent and 47% with the other one; in contrast, the putative parental stocks shared less than 30% of the mHVR clusters between them. CONCLUSIONS/SIGNIFICANCE: The results suggest a reductive division, a natural hybridization, biparental inheritance of the minicircles in the hybrid and maxicircle introgression. The models including such phenomena and explaining the relationships between these three clones are discussed.


Assuntos
DNA de Protozoário/genética , Hibridização Genética , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , DNA de Cinetoplasto/genética , Genes de Protozoários , Sequenciamento de Nucleotídeos em Larga Escala , Ploidias , Análise de Sequência de DNA
5.
BMC Vet Res ; 15(1): 344, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619238

RESUMO

BACKGROUND: African animal trypanosomosis remains the major constraint of livestock production and livelihood of pastoral communities in Cameroon. Despite several decades of vector and parasite control efforts, it has not been eradicated. Alternative and sustainable control strategies require a sound knowledge of the local species, strains and vectors. In the Sudano-Sahelian and Guinea Savannah of Cameroon the prevalence and genetic diversity of trypanosomes infecting cattle was investigated by microscopy of cattle blood buffy coat and molecular methods using generic primers targeting parts of the internal transcribed spacer 1 (ITS-1) and encoded glycosomal glyceraldehyde 3-phosphate dehydrogenase-gene (gGAPDH). RESULTS: A total of 1176 randomly chosen cattle from five divisions in the Sudano-Sahelian and Guinea Savannah of Cameroon were examined. The overall prevalence of trypanosomes by microscopy was 5.9% (56/953) in contrast to 53.2% (626/1176) when molecular tools were used. This indicated a limited sensitivity of microscopy in subclinical infections with frequently low parasitemia. Three trypanosome species were identified by light microscopy: T. vivax (2.3%), T. brucei (3.7%) and T. congolense (3.0%), whereas five were identified by PCR, namely T. grayi/T. theileri (30.8%), T. vivax (17.7%), T. brucei (14.5%) and T. congolense (5.1%). Unexpected cases of T. grayi (n = 4) and T. theileri (n = 26) were confirmed by sequencing. Phylogenetic analysis of the gGAPDH revealed the presence of T. vivax, clade A and T. vivax clade C, which were co-endemic in the Faro et Deo division. T. grayi/T. theileri were the predominant species infecting cattle in tsetse free areas. In contrast, T. vivax, T. brucei and T. congolense were more abundant in areas where the Glossina-vectors were present. CONCLUSIONS: The abundance of pathogenic trypanosomes in tsetse infested areas is alarming and even more, the occurrence of T. vivax, T. brucei, T. congolense, T. theileri and T. grayi in tsetse-free areas implies that tsetse control alone is not sufficient to control trypanosomosis in livestock. To implement control measures that reduce the risk of spread in tsetse free areas, close monitoring using molecular tools and a thorough search for alternative vectors of trypanosomes is recommended.


Assuntos
Doenças dos Bovinos/epidemiologia , Trypanosoma/isolamento & purificação , Tripanossomíase Africana/epidemiologia , Animais , Buffy Coat/parasitologia , Camarões/epidemiologia , Bovinos , Doenças dos Bovinos/parasitologia , Feminino , Genes de Protozoários , Insetos Vetores , Masculino , Prevalência , Trypanosoma/classificação , Trypanosoma/genética , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/prevenção & controle , Moscas Tsé-Tsé
6.
PLoS Negl Trop Dis ; 13(10): e0007750, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31661498

RESUMO

BACKGROUND: The World Health Organization WHO has estimated that in developed countries, up to 30% of the population may suffer from foodborne diseases each year, and that in developing countries up to 2 million deaths per annum can be attributed to cryptosporidiosis. Reports have already emphasized the role of immigrants in outbreaks of parasitic diseases especially those working in food processing industries. METHODOLOGY/PRINCIPAL FINDINGS: Herein we assessed Cryptosporidium spp. infections among immigrants in Qatar with a special focus on food handlers and housemaids. The overall prevalence of Cryptosporidium spp. by q-PCR among 839 asymptomatic subjects was 4.5%. Based on the Gp60 gene, the majority of isolates were identified as C. parvum subtype IIdA20G1b. The positive sample for C. hominis was subtyped as IeA12G3T3. Seven mixed infections were also identified (four C. parvum + C. hominis, and three C. parvum + C. meleagridis). The prevalence of Cryptosporidium spp. did not differ significantly between the sexes or age classes but varied significantly between subjects affiliated to different religions with the lowest prevalence among the Muslims. Multifactorial analysis retained also marked significance with education, income, and a house contents index. CONCLUSIONS/SIGNIFICANCE: Our results contribute to a better understanding of the epidemiology of cryptosporidiosis and the risk factors associated with the likelihood of carrying this infection among immigrant workers from developing countries.


Assuntos
Criptosporidiose/epidemiologia , Cryptosporidium/isolamento & purificação , Migrantes , Adolescente , Adulto , Criptosporidiose/diagnóstico , Cryptosporidium/genética , DNA de Protozoário/genética , Surtos de Doenças , Feminino , Genes de Protozoários , Genótipo , Técnicas de Genotipagem , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Patologia Molecular , Prevalência , Catar/epidemiologia , Fatores de Risco , Adulto Jovem
7.
PLoS Pathog ; 15(9): e1008065, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31557263

RESUMO

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.


Assuntos
Proteínas de Protozoários/metabolismo , Tiorredoxinas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Técnicas de Silenciamento de Genes , Genes de Protozoários , Humanos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Oxirredução , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/patogenicidade
8.
PLoS Comput Biol ; 15(9): e1007329, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31509524

RESUMO

Empirical evidence suggests that the malaria parasite Plasmodium falciparum employs a broad range of mechanisms to regulate gene transcription throughout the organism's complex life cycle. To better understand this regulatory machinery, we assembled a rich collection of genomic and epigenomic data sets, including information about transcription factor (TF) binding motifs, patterns of covalent histone modifications, nucleosome occupancy, GC content, and global 3D genome architecture. We used these data to train machine learning models to discriminate between high-expression and low-expression genes, focusing on three distinct stages of the red blood cell phase of the Plasmodium life cycle. Our results highlight the importance of histone modifications and 3D chromatin architecture in Plasmodium transcriptional regulation and suggest that AP2 transcription factors may play a limited regulatory role, perhaps operating in conjunction with epigenetic factors.


Assuntos
Biologia Computacional/métodos , Código das Histonas/genética , Modelos Estatísticos , Nucleossomos/genética , Plasmodium falciparum/genética , Eritrócitos/parasitologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Genes de Protozoários/genética , Humanos , Estágios do Ciclo de Vida/genética , Aprendizado de Máquina , Malária Falciparum , Modelos Biológicos , Plasmodium falciparum/citologia , Plasmodium falciparum/patogenicidade
9.
Parasitol Res ; 118(10): 3043-3051, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31471745

RESUMO

Using a combination of morphological and molecular data, we describe a new apicomplexan parasite, Isospora svecica sp. n., from the white-spotted bluethroat, Luscinia svecica cyanecula, from the Czech Republic. Oocysts were found in its intestinal tract. Sporulation was exogenous and took 1-3 days. The oocysts were slightly ellipsoidal, of average size 26.17 × 20.33 µm, with a smooth bilayered wall. Micropyle, oocyst residuum, and polar granules were absent. Sporocysts were bottle-shaped, of an average size of 18.82 × 8.82 µm, with a thin, colourless wall. A conspicuous knob-like Stieda body was present. Substieda body was barely visible. Sporocyst residuum was present in the form of granules of various sizes. Sporozoites were banana-shaped and contained large anterior and small posterior refractile bodies. Partial DNA sequences of three genes were obtained from oocysts of Isospora svecica sp. n., being most closely related to other isosporans described from passerines. Little is known about the parasites of the avian family Muscicapidae, including coccidia, a highly prevalent parasitic protist group in all vertebrate classes. Only six species of the genus Isospora have so far been described in Muscicapidae, together with several "Isospora sp." that in fact most likely represent Isospora lacazei. The newly described Isospora svecica sp. n. differs morphologically from other coccidia reported from muscicapid birds, and represents the first coccidian species described from Luscinia svecica.


Assuntos
Isospora/classificação , Isosporíase/veterinária , Passeriformes/parasitologia , Animais , República Tcheca , Genes de Protozoários/genética , Intestinos/parasitologia , Isospora/citologia , Isospora/genética , Isospora/crescimento & desenvolvimento , Isosporíase/parasitologia , Oocistos/classificação , Oocistos/citologia , Oocistos/genética , Oocistos/crescimento & desenvolvimento , Esporozoítos/classificação , Esporozoítos/citologia , Esporozoítos/genética , Esporozoítos/crescimento & desenvolvimento
10.
Nat Commun ; 10(1): 4300, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541097

RESUMO

Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Cruzamentos Genéticos , Resistência a Medicamentos/genética , Proteínas de Membrana Transportadoras/genética , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Animais , Anopheles/parasitologia , Culicidae/parasitologia , Descoberta de Drogas , Feminino , Expressão Gênica , Genes de Protozoários , Malária/tratamento farmacológico , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Masculino , Plasmodium falciparum/genética
11.
Science ; 365(6455)2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31439762

RESUMO

Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We profiled the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then used our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a eukaryotic parasite and an open-access reference dataset for the study of malaria parasites.


Assuntos
Atlas como Assunto , Genes de Protozoários/fisiologia , Estágios do Ciclo de Vida/genética , Malária/parasitologia , Plasmodium berghei/genética , Plasmodium berghei/fisiologia , Transcriptoma , Animais , Anopheles/parasitologia , Células HeLa , Humanos , Plasmodium berghei/isolamento & purificação , Análise de Célula Única
12.
Vet Parasitol ; 273: 32-35, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31442890

RESUMO

This study looked to assess the stability of Cryptosporidium parvum genotypes in calves between the final day of treatment with the antiprotozoal halofuginone lactate and seven days post-treatment. Paired faecal samples were collected on the final day of treatment and seven days later from 54 calves across seven farms in South-west England. The presence of Cryptosporidium species was detected using polymerase chain reaction targeting the 18 s rDNA. The presence and genotype of C. parvum was determined by PCR and amplicon sequencing targeting the gp60 locus. On farms where C. parvum was detected at both sampling times there was a distinct genotype shift. Detection of gp60 genotype IIaA15G2R1 decreased from 40% to 7% while IIaA17G1R1 increased from 0% to 41%, supplemented by IIaA16G3R1 in one sample. A shift in C. parvum genotypes present in calves within a one week sampling timeframe has not been described prior to this study, indicating that the timeframe is likely suitable for observing variation in C. parvum populations and interactions with antiprotozoal control strategies.


Assuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , Animais , Antiprotozoários/uso terapêutico , Bovinos , Doenças dos Bovinos/tratamento farmacológico , Criptosporidiose/tratamento farmacológico , Inglaterra , Fezes/parasitologia , Genes de Protozoários/genética , Genótipo , RNA Ribossômico 18S/genética
13.
Parasite ; 26: 53, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31448999

RESUMO

To study the presence of Giardia duodenalis in Xinjiang, northwest China, we collected 801 fecal specimens from seven large-scale pig farms and screened them using PCR targeting the SSU rRNA gene. Twenty-one (2.6%) of the specimens from five farms were G. duodenalis-positive, with a significant difference in prevalence among different farms (0-8.7%) (p < 0.01). Giardia duodenalis prevalence was highest in fattening pigs (5.4%, 7/129), followed by sows (3.2%, 7/222), post-weaning piglets (1.8%, 5/281), and pre-weaning piglets (1.2%, 2/169), but there was no significant difference in prevalence among the age groups (p > 0.05). Sequence analysis of the SSU rRNA gene revealed that the 21 G. duodenalis strains belonged to three assemblages: A (n = 2), B (n = 16), and E (n = 3). Assemblage B was the predominant assemblage and was widely distributed in all G. duodenalis-positive farms and age groups. All G. duodenalis-positive specimens were further assayed at the ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes, and two tpi, four gdh, and two bg sequences were identified. These data indicate that pigs may be a zoonotic risk and can potentially spread G. duodenalis infection from animals to humans.


Assuntos
Giardia lamblia/genética , Giardia lamblia/isolamento & purificação , Giardíase/veterinária , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/parasitologia , Animais , China/epidemiologia , Fazendas , Fezes/parasitologia , Feminino , Genes de Protozoários , Genótipo , Giardíase/epidemiologia , Giardíase/parasitologia , Masculino , Prevalência , Proteínas de Protozoários/genética , Suínos/parasitologia
14.
PLoS Pathog ; 15(7): e1007973, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31348803

RESUMO

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle.


Assuntos
Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/fisiologia , Proteína Fosfatase 1/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Anopheles/parasitologia , Eritrócitos/parasitologia , Feminino , Genes de Protozoários , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Humanos , Malária/parasitologia , Malária/transmissão , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mosquitos Vetores/parasitologia , Plasmodium berghei/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
PLoS Pathog ; 15(7): e1007946, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31348812

RESUMO

By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.


Assuntos
Proteínas F-Box/fisiologia , Proteínas de Protozoários/fisiologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade , Animais , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/genética , Técnicas de Silenciamento de Genes , Genes de Protozoários , Humanos , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas Quinases Associadas a Fase S/fisiologia , Toxoplasma/genética
16.
PLoS Negl Trop Dis ; 13(7): e0007570, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31356610

RESUMO

Kinetoplastids are a group of parasites that includes several medically-important species. These human-infective species are transmitted by insect vectors in which the parasites undergo specific developmental transformations. For each species, this includes a stage in which parasites adhere to insect tissue via a hemidesmosome-like structure. Although this structure has been described morphologically, it has never been molecularly characterized. We are using Crithidia fasciculata, an insect parasite that produces large numbers of adherent parasites inside its mosquito host, as a model kinetoplastid to investigate both the mechanism of adherence and the signals required for differentiation to an adherent form. An advantage of C. fasciculata is that adherent parasites can be generated both in vitro, allowing a direct comparison to cultured swimming forms, as well as in vivo within the mosquito. Using RNAseq, we identify genes associated with adherence in C. fasciculata. As almost all of these genes have orthologs in other kinetoplastid species, our findings may reveal shared mechanisms of adherence, allowing investigation of a crucial step in parasite development and disease transmission. In addition, dual-RNAseq allowed us to explore the interaction between the parasites and the mosquito. Although the infection is well-tolerated, anti-microbial peptides and other components of the mosquito innate immune system are upregulated. Our findings indicate that C. fasciculata is a powerful model system for probing kinetoplastid-insect interactions.


Assuntos
Aedes/parasitologia , Crithidia fasciculata/genética , Genes de Protozoários , Aedes/anatomia & histologia , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Crithidia fasciculata/crescimento & desenvolvimento , Crithidia fasciculata/fisiologia , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita , Masculino , RNA de Protozoário , Análise de Sequência de RNA , Transdução de Sinais
17.
Pathog Glob Health ; 113(4): 158-166, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31296112

RESUMO

Malaria remains a significant public health challenge and is of global importance. Imported malaria is a growing problem in non-endemic areas throughout the world and also in Qatar due to a massive influx of migrants from endemic countries. Antimalarial drug resistance is an important deterrent in our fight against malaria today. Molecular markers mirror intrinsic antimalarial drug resistance and their changes precede clinical resistance. Thus, in the present study, molecular markers of sulphadoxine-pyrimethamine (Pfdhfr and Pfdhps) and artemisinin (PfATPase6 and Pfk13) were sequenced to determine the drug resistance genotypes among 118 imported P. falciparum isolates in Qatar, between 2013 and 2016. All the isolates had mutant Pfdhfr alleles, with either double mutant (51I/108N) (59.3%) or triple mutant (51I, 59R and 108N) (30.6%) genotypes. I164L substitution was not found in this study. In case of Pfdhps, majority of the samples were carriers of either single (S436A/ A437G/ K540E) mutant (47.2%) or double (S436A/K540E, A437G/K540E, K540E/A581G) mutant (39.8%). A single novel point mutation (431V) was observed in the samples originated from Nigeria and Ghana. Polymorphisms in PfATPase6 were absent and only one non-synonymous mutation in Pfk13 was found at codon G453A from a sample of Kenyan origin. High levels of sulphadoxine-pyrimethamine resistance in the present study provide potential information about the spread of antimalarial drug resistance and will be beneficial for the treatment of imported malaria cases in Qatar.


Assuntos
Antiprotozoários/farmacologia , Artemisininas/farmacologia , Doenças Transmissíveis Importadas/parasitologia , Resistência a Medicamentos , Lactonas/farmacologia , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Pirimetamina/farmacologia , Sulfadoxina/farmacologia , Adulto , Doenças Transmissíveis Importadas/epidemiologia , Combinação de Medicamentos , Monitoramento Epidemiológico , Feminino , Genes de Protozoários , Genótipo , Humanos , Malária Falciparum/epidemiologia , Masculino , Epidemiologia Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/isolamento & purificação , Catar/epidemiologia , Análise de Sequência de DNA
18.
Genes (Basel) ; 10(7)2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315304

RESUMO

Malaria is a significant public health problem in Ghana. Seasonal Malaria Chemoprevention (SMC) using a combination of sulfadoxine-pyrimethamine and amodiaquine has been implemented since 2015 in northern Ghana where malaria transmission is intense and seasonal. In this study, we estimated the prevalence of asymptomatic P. falciparum carriers in three ecological zones of Ghana, and compared the sensitivity and specificity of different molecular methods in identifying asymptomatic infections. Moreover, we examined the frequency of mutations in pfcrt, pfmdr1, pfdhfr, and pfdhps that relate to the ongoing SMC. A total of 535 asymptomatic schoolchildren were screened by microscopy and PCR (18s rRNA and TARE-2) methods. Among all samples, 28.6% were detected as positive by 18S nested PCR, whereas 19.6% were detected by microscopy. A high PCR-based asymptomatic prevalence was observed in the north (51%) compared to in the central (27.8%) and south (16.9%). The prevalence of pfdhfr-N51I/C59R/S108N/pfdhps-A437G quadruple mutant associated with sulfadoxine-pyrimethamine resistance was significantly higher in the north where SMC was implemented. Compared to 18S rRNA, TARE-2 serves as a more sensitive molecular marker for detecting submicroscopic asymptomatic infections in high and low transmission settings. These findings establish a baseline for monitoring P. falciparum prevalence and resistance in response to SMC over time.


Assuntos
Resistência a Medicamentos , Genes de Protozoários , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Estações do Ano , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Doenças Assintomáticas , Quimioprevenção , Gana/epidemiologia , Haplótipos , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/prevenção & controle , Mutação , Prevalência , Vigilância em Saúde Pública , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade
19.
PLoS Pathog ; 15(7): e1007906, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31295334

RESUMO

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host's immune system, thus promoting overall parasite survival in the face of host immunity.


Assuntos
Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium falciparum/patogenicidade , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Feminino , Regulação da Expressão Gênica , Genes de Protozoários , Humanos , Imunidade Inata , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Virulência/genética , Virulência/imunologia , Adulto Jovem
20.
Genes (Basel) ; 10(7)2019 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-31284605

RESUMO

A conspicuous cell-shape phenotype known as "screwy" was reported to result from mutations at two or three uncharacterized loci in the ciliate Paramecium tetraurelia. Here, we describe a new screwy mutation, Spinning Top, which appeared spontaneously in the cross of an unrelated mutant with reference strain 51. The macronuclear (MAC) genome of the Spinning Top mutant is shown to lack a ~28.5-kb segment containing 18 genes at the end of one chromosome, which appears to result from a collinear deletion in the micronuclear (MIC) genome. We tested several candidate genes from the deleted locus by dsRNA-induced silencing in wild-type cells, and identified a single gene responsible for the phenotype. This gene, named Spade, encodes a 566-aa glutamine-rich protein with a C2HC zinc finger. Its silencing leads to a fast phenotype switch during vegetative growth, but cells recover a wild-type phenotype only 5-6 divisions after silencing is stopped. We analyzed 5 independently-obtained mutant alleles of the Sc1 locus, and concluded that all of them also lack the Spade gene and a number of neighboring genes in the MAC and MIC genomes. Mapping of the MAC deletion breakpoints revealed two different positions among the 5 alleles, both of which differ from the Spinning Top breakpoint. These results suggest that this MIC chromosome region is intrinsically unstable in strain 51.


Assuntos
Genes de Protozoários , Paramecium tetraurellia/genética , Proteínas de Protozoários/genética , Fragilidade Cromossômica , Mutação , Fenótipo
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