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1.
PLoS Genet ; 16(11): e1009101, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33196661

RESUMO

Characterising connectivity between geographically separated biological populations is a common goal in many fields. Recent approaches to understanding connectivity between malaria parasite populations, with implications for disease control efforts, have used estimates of relatedness based on identity-by-descent (IBD). However, uncertainty around estimated relatedness has not been accounted for. IBD-based relatedness estimates with uncertainty were computed for pairs of monoclonal Plasmodium falciparum samples collected from five cities on the Colombian-Pacific coast where long-term clonal propagation of P. falciparum is frequent. The cities include two official ports, Buenaventura and Tumaco, that are separated geographically but connected by frequent marine traffic. Fractions of highly-related sample pairs (whose classification using a threshold accounts for uncertainty) were greater within cities versus between. However, based on both highly-related fractions and on a threshold-free approach (Wasserstein distances between parasite populations) connectivity between Buenaventura and Tumaco was disproportionally high. Buenaventura-Tumaco connectivity was consistent with transmission events involving parasites from five clonal components (groups of statistically indistinguishable parasites identified under a graph theoretic framework). To conclude, P. falciparum population connectivity on the Colombian-Pacific coast abides by accessibility not isolation-by-distance, potentially implicating marine traffic in malaria transmission with opportunities for targeted intervention. Further investigations are required to test this hypothesis. For the first time in malaria epidemiology (and to our knowledge in ecological and epidemiological studies more generally), we account for uncertainty around estimated relatedness (an important consideration for studies that plan to use genotype versus whole genome sequence data to estimate IBD-based relatedness); we also use threshold-free methods to compare parasite populations and identify clonal components. Threshold-free methods are especially important in analyses of malaria parasites and other recombining organisms with mixed mating systems where thresholds do not have clear interpretation (e.g. due to clonal propagation) and thus undermine the cross-comparison of studies.


Assuntos
Genoma de Protozoário/genética , Malária Falciparum/parasitologia , Modelos Genéticos , Plasmodium falciparum/genética , Colômbia/epidemiologia , Frequência do Gene , Técnicas de Genotipagem , Humanos , Malária Falciparum/epidemiologia , Malária Falciparum/transmissão , Cadeias de Markov , Plasmodium falciparum/isolamento & purificação , Polimorfismo de Nucleotídeo Único , Reprodução Assexuada/genética , Análise Espaço-Temporal , Incerteza
2.
PLoS Negl Trop Dis ; 14(10): e0008234, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33044985

RESUMO

Plasmodium vivax malaria is much less common in Africa than the rest of the world because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a high number of them being in Duffy negative individuals, potentially indicating P. vivax has evolved an alternative invasion mechanism that can overcome Duffy negativity. Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and Duffy negative individuals are found. A total of 123,711 SNPs were detected, of which 22.7% were nonsynonymous and 77.3% were synonymous mutations. The largest number of SNPs were detected on chromosomes 9 (24,007 SNPs; 19.4% of total) and 10 (16,852 SNPs, 13.6% of total). There were particularly high levels of polymorphism in erythrocyte binding gene candidates including merozoite surface protein 1 (MSP1) and merozoite surface protein 3 (MSP3.5, MSP3.85 and MSP3.9). Two genes, MAEBL and MSP3.8 related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. Variation in gene copy number was also concentrated in genes involved in host-parasite interactions, including the expansion of the Duffy binding protein gene (PvDBP) on chromosome 6 and MSP3.11 on chromosome 10. Based on the phylogeny constructed from the whole genome sequences, the expansion of these genes was an independent process among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. vivax infections among study sites and showed various levels of gene flow at a small geographical scale. The genomic features of P. vivax provided baseline data for future comparison with those in Duffy-negative individuals and allowed us to develop a panel of informative Single Nucleotide Polymorphic markers diagnostic at a micro-geographical scale.


Assuntos
Malária Vivax/parasitologia , Plasmodium vivax/genética , Sequenciamento Completo do Genoma , Antígenos de Protozoários/genética , Variações do Número de Cópias de DNA , DNA de Protozoário , Sistema do Grupo Sanguíneo Duffy/genética , Eritrócitos/parasitologia , Etiópia , Genoma de Protozoário , Humanos , Malária Vivax/genética , Filogenia , Plasmodium vivax/classificação , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Receptores de Superfície Celular/genética
3.
Exp Parasitol ; 219: 108015, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33031787

RESUMO

Lack of knowledge of taxonomic biodiversity and reliable genetic markers in Trypanosomatidae limit our understanding of their phylogenetic relationships. Ultraconserved elements (UCEs) have improved phylogenetic analyses and inferences in many vertebrate and invertebrate taxa. However, it is unknown whether protozoans have these markers, their abundance, and if these could be reliably used for phylogenetics. In this study I design a target enrichment bait set for UCE loci for this group. In silico testing showed good loci recovery rates across 63 taxa and produced consistent, highly supported phylogenetic trees. This bait set adds a new resource of useful genetic markers for Trypanosomatidae phylogenetics.


Assuntos
Marcadores Genéticos , Filogenia , Trypanosomatina/classificação , Trypanosomatina/genética , Genoma de Protozoário
4.
Nat Commun ; 11(1): 5258, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067458

RESUMO

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.


Assuntos
Interferon gama/imunologia , Macrófagos/imunologia , Toxoplasma/genética , Toxoplasmose/imunologia , Animais , Feminino , Genoma de Protozoário , Interações Hospedeiro-Parasita , Humanos , Interferon gama/genética , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/genética , Toxoplasmose/parasitologia , Virulência
5.
Protist ; 171(5): 125758, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33126018

RESUMO

Coelastrum proboscideum Bohlin, 1896 (Sphaeropleales, Scenedesmaceae, Chlorophyta) is a coenobial species with cosmopolitan distribution in diverse freshwater habitats. Coelastrum spp. are widely tested for biotechnological applications such as carotenoid and lipid production, and in bioremediation of wastewater. Here, we report the draft genome of C. proboscideum var. dilatatum strain SAG 217-2. The final assembly comprised 125,935,854 bp with over 8357 scaffolds. The whole-genome data is publicly available in the Nucleotide Sequence Archive (CNSA) of China National GeneBank (CNGB) (https://db.cngb.org/cnsa/) under the accession number CNA0014153.


Assuntos
Clorófitas/genética , Genoma de Protozoário , Queixo , Bases de Dados Genéticas
6.
Sci Rep ; 10(1): 15043, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929126

RESUMO

Here, we report a pilot study paving the way for further single cell genomics studies in Leishmania. First, the performances of two commercially available kits for Whole Genome Amplification (WGA), PicoPLEX and RepliG were compared on small amounts of Leishmania donovani DNA, testing their ability to preserve specific genetic variations, including aneuploidy levels and SNPs. We show here that the choice of WGA method should be determined by the planned downstream genetic analysis, PicoPLEX and RepliG performing better for aneuploidy and SNP calling, respectively. This comparison allowed us to evaluate and optimize corresponding bio-informatic methods. As PicoPLEX was shown to be the preferred method for studying single cell aneuploidy, this method was applied in a second step, on single cells of L. braziliensis, which were sorted by fluorescence activated cell sorting (FACS). Even sequencing depth was achieved in 28 single cells, allowing accurate somy estimation. A dominant karyotype with three aneuploid chromosomes was observed in 25 cells, while two different minor karyotypes were observed in the other cells. Our method thus allowed the detection of aneuploidy mosaicism, and provides a solid basis which can be further refined to concur with higher-throughput single cell genomic methods.


Assuntos
Biologia Computacional/métodos , Genoma de Protozoário , Cariotipagem/métodos , Leishmania/genética , Análise de Célula Única/métodos , Aneuploidia , Citometria de Fluxo/métodos
7.
PLoS Negl Trop Dis ; 14(9): e0008684, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946436

RESUMO

Leishmania tropica is one of the main causative agents of cutaneous leishmaniasis (CL). Population structures of L. tropica appear to be genetically highly diverse. However, the relationship between L. tropica strains genomic diversity, protein coding gene evolution and biogeography are still poorly understood. In this study, we sequenced the genomes of three new clinical L. tropica isolates, two derived from a recent outbreak of CL in camps hosting Syrian refugees in Lebanon and one historical isolate from Azerbaijan to further refine comparative genome analyses. In silico multilocus microsatellite typing (MLMT) was performed to integrate the current diversity of genome sequence data in the wider available MLMT genetic population framework. Single nucleotide polymorphism (SNPs), gene copy number variations (CNVs) and chromosome ploidy were investigated across the available 18 L. tropica genomes with a main focus on protein coding genes. MLMT divided the strains in three populations that broadly correlated with their geographical distribution but not populations defined by SNPs. Unique SNPs profiles divided the 18 strains into five populations based on principal component analysis. Gene ontology enrichment analysis of the protein coding genes with population specific SNPs profiles revealed various biological processes, including iron acquisition, sterols synthesis and drug resistance. This study further highlights the complex links between L. tropica important genomic heterogeneity and the parasite broad geographic distribution. Unique sequence features in protein coding genes identified in distinct populations reveal potential novel markers that could be exploited for the development of more accurate typing schemes to further improve our knowledge of the evolution and epidemiology of the parasite as well as highlighting protein variants of potential functional importance underlying L. tropica specific biology.


Assuntos
Variação Genética , Genoma de Protozoário , Leishmania tropica/genética , Azerbaijão , Variações do Número de Cópias de DNA , DNA de Protozoário/genética , Dosagem de Genes , Mapeamento Geográfico , Humanos , Líbano , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Repetições de Microssatélites , Filogenia , Polimorfismo de Nucleotídeo Único , Refugiados , Síria
8.
Nucleic Acids Res ; 48(17): 9660-9680, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890403

RESUMO

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.


Assuntos
Variação Antigênica , DNA Polimerase Dirigida por DNA/metabolismo , Telômero/genética , Trypanosoma brucei brucei/genética , Linhagem Celular , Núcleo Celular/enzimologia , Núcleo Celular/genética , Segregação de Cromossomos , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Regulação da Expressão Gênica , Genoma de Protozoário , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , Telômero/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/genética
9.
PLoS Pathog ; 16(8): e1008772, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866214

RESUMO

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.


Assuntos
Antígenos de Protozoários , Babesia bovis , Conversão Gênica/imunologia , Genoma de Protozoário/imunologia , Proteínas de Protozoários , Rad51 Recombinase , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Babesia bovis/genética , Babesia bovis/imunologia , DNA de Protozoário/genética , DNA de Protozoário/imunologia , Haploidia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Rad51 Recombinase/genética , Rad51 Recombinase/imunologia
10.
Trends Parasitol ; 36(11): 927-941, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32828660

RESUMO

Lateral gene transfer (LGT) is well known as an important driver of genome evolution in bacteria and archaea, but its importance in eukaryote evolution has yet to be fully elucidated. There is now abundant evidence indicating that LGT has played a role in the adaptation of eukaryotes to new environments and conditions, including host-parasite interactions. However, the mechanisms and frequency of LGT across the tree of eukaryotes remain poorly understood. Here we review evidence for known and potential mechanisms of LGT into diverse eukaryote lineages with a particular focus on protists, and we discuss trends emerging from recently reported examples. We also explore the potential role of LGT in generating 'pan-genomes' in diverse eukaryotic species.


Assuntos
Eucariotos/genética , Transferência Genética Horizontal/genética , Genoma/genética , Evolução Molecular , Genoma de Protozoário/genética , Interações Hospedeiro-Parasita/genética
11.
PLoS One ; 15(8): e0237180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750094

RESUMO

BACKGROUND: Chagas disease, caused by the intracellular parasite Trypanosoma cruzi, is one of the most important parasitological infections in the Americas. It is estimated to infect approximately 6 million people from mostly low income countries in Latin America, although recent infections have been reported in southern US states. Several studies have described an extensive genetic diversity among T. cruzi isolates throughout its geographic distribution in the American continent. This diversity has been correlated with the pathology developed during an infection. However, due to a lack of a single reliable test, current diagnosis practices of the disease are not straightforward since several different tests are applied. The use of current genomic sequence data allows for the selection of molecular markers (MM) that have the ability to identify the Discrete Typing Unit (DTU) of T. cruzi in a given infection, without the need of any sequencing reaction. METHODOLOGY/PRINCIPAL FINDINGS: Applying three criteria on the genomic sequencing data of four different phylogenetic lineages of T. cruzi, we designed several molecular tests that can be used for the molecular typing of the parasite. The criteria used were: (1) single-copy orthologs of T. cruzi, (2) T. cruzi unique loci, and (3) T. cruzi polymorphic loci. All criteria combined allowed for the selection of 15 MM, 12 of which were confirmed to be functional and replicable in the laboratory with sylvatic samples. Furthermore, one MM produced distinct polymerase chain reaction (PCR) amplicon sizes among distinct T. cruzi DTUs, allowing the use of a AFLP-PCR test to distinguish DTUs I, II/IV, V and VI. Whereas two MM can differentiate DTUs I, II, IV and V/VI out of the six current DTUs with a PCR-RFLP test. CONCLUSIONS/SIGNIFICANCE: The designed molecular tests provide a practical and inexpensive molecular typing test for the majority of DTUs of T. cruzi, excluding the need to perform any sequencing reaction. This provides the scientific community with an additional specific, quick and inexpensive test that can enhance the understanding of the correlation between the DTU of T. cruzi and the pathology developed during the infection.


Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Doença de Chagas/diagnóstico , Polimorfismo de Fragmento de Restrição/genética , Trypanosoma cruzi/genética , Doença de Chagas/parasitologia , DNA de Protozoário/genética , Loci Gênicos , Variação Genética , Genoma de Protozoário/genética , Humanos , Tipagem Molecular/métodos , Filogenia , Polimorfismo de Nucleotídeo Único
12.
PLoS Pathog ; 16(8): e1008717, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745123

RESUMO

Hepatocystis is a genus of single-celled parasites infecting, amongst other hosts, monkeys, bats and squirrels. Although thought to have descended from malaria parasites (Plasmodium spp.), Hepatocystis spp. are thought not to undergo replication in the blood-the part of the Plasmodium life cycle which causes the symptoms of malaria. Furthermore, Hepatocystis is transmitted by biting midges, not mosquitoes. Comparative genomics of Hepatocystis and Plasmodium species therefore presents an opportunity to better understand some of the most important aspects of malaria parasite biology. We were able to generate a draft genome for Hepatocystis sp. using DNA sequencing reads from the blood of a naturally infected red colobus monkey. We provide robust phylogenetic support for Hepatocystis sp. as a sister group to Plasmodium parasites infecting rodents. We show transcriptomic support for a lack of replication in the blood and genomic support for a complete loss of a family of genes involved in red blood cell invasion. Our analyses highlight the rapid evolution of genes involved in parasite vector stages, revealing genes that may be critical for interactions between malaria parasites and mosquitoes.


Assuntos
Apicomplexa/genética , Sangue/parasitologia , Colobus/parasitologia , Malária/veterinária , Doenças dos Macacos/parasitologia , Plasmodium/genética , Infecções Protozoárias em Animais/parasitologia , Animais , Apicomplexa/classificação , Apicomplexa/fisiologia , Genoma de Protozoário , Malária/sangue , Malária/parasitologia , Doenças dos Macacos/sangue , Filogenia , Plasmodium/classificação , Plasmodium/fisiologia , Infecções Protozoárias em Animais/sangue , Transcriptoma
13.
Eur J Protistol ; 76: 125722, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32679518

RESUMO

Cilia and flagella play an important role in motility, sensory perception, and the life cycles of eukaryotes, from protists to humans. However, much critical information concerning cilia structure and function remains elusive. The vast majority of ciliary and flagellar proteins analyzed so far are evolutionarily conserved and play a similar role in protozoa and vertebrates. This makes protozoa attractive biological models for studying cilia biology. Research conducted on ciliated or flagellated protists may improve our general understanding of cilia protein composition, of cilia beating, and can shed light on the molecular basis of the human disorders caused by motile cilia dysfunction. The Symposium "From genomics to flagellar and ciliary structures and cytoskeleton dynamics" at ECOP2019 in Rome presented the latest discoveries about cilia biogenesis and the molecular mechanisms of ciliary and flagellum motility based on studies in Paramecium, Tetrahymena, and Trypanosoma. Here, we review the most relevant aspects presented and discussed during the symposium and add our perspectives for future research.


Assuntos
Citoesqueleto/ultraestrutura , Genoma de Protozoário/genética , Paramecium , Tetrahymena , Trypanosoma , Cílios/genética , Congressos como Assunto , Flagelos/genética , Paramecium/genética , Paramecium/ultraestrutura , Tetrahymena/genética , Tetrahymena/ultraestrutura , Trypanosoma/genética , Trypanosoma/ultraestrutura
14.
Sci Rep ; 10(1): 12321, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32704096

RESUMO

Visceral leishmaniasis (VL) is a life-threatening disease caused by the protozoa Leishmania donovani and L. infantum. Likely, L. infantum was introduced in the New World by the Iberic colonizers. Due to recent introduction, the genetic diversity is low. Access to genomic information through the sequencing of Leishmania isolates allows the characterization of populations through the identification and analysis of variations. Population structure information may reveal important data on disease dynamics. Aiming to describe the genetic diversity of L. infantum from the Middle-North, Brazil, next generation sequencing of 30 Leishmania isolates obtained in the city of Teresina, from where the disease dispersed, was performed. The variations were categorized accordingly to the genome region and impact and provided the basis for chromosomal ploidy and population structure analysis. The results showed low diversity between the isolates and the Iberic reference genome JPCM5. Most variations were seen in non-coding regions, with modifying impact. The ploidy number analysis showed aneuploid profile. The population structure analysis revealed the presence of two L. infantum populations identified in Teresina. Further population genetics studies with a larger number of isolates should be performed in order to identify the genetic background associated with virulence and parasite ecology.


Assuntos
Genoma de Protozoário , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leishmania infantum/genética , Brasil , Cromossomos/genética , Variações do Número de Cópias de DNA/genética , Leishmania infantum/isolamento & purificação , Funções Verossimilhança , Filogenia , Polimorfismo de Nucleotídeo Único/genética
15.
Sci Rep ; 10(1): 11157, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636411

RESUMO

The DNA polymerase module of the Pfprex enzyme (PfpPol) is responsible for duplication of the genome of the apicoplast organelle in the malaria parasite. We show that PfpPol can misincorporate oxidized nucleotides such as 8oxodGTP opposite dA. This event gives rise to transversion mutations that are known to lead to adverse physiological outcomes. The apicoplast genome is particularly vulnerable to the harmful effects of 8oxodGTP due to very high AT content (~ 87%). We show that the proofreading activity of PfpPol has the unique ability to remove the oxidized nucleotide from the primer terminus. Due to this property, the proofreading domain of PfpPol is able to prevent mutagenesis of the AT-rich apicoplast genome and neutralize the deleterious genotoxic effects of ROS generated in the apicoplast due to normal metabolic processes. The proofreading activity of the Pfprex enzyme may, therefore, represent an attractive target for therapeutic intervention. Also, a survey of DNA repair pathways shows that the observed property of Pfprex constitutes a novel form of dynamic error correction wherein the repair of promutagenic damaged nucleotides is concomitant with DNA replication.


Assuntos
Apicoplastos/metabolismo , Reparo do DNA , Nucleotídeos de Desoxiguanina/metabolismo , Complexos Multienzimáticos/fisiologia , Mutagênese/genética , Nucleotídeos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/fisiologia , Apicoplastos/genética , Genoma de Protozoário/genética , Complexos Multienzimáticos/metabolismo , Oxirredução , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo
16.
Sci Rep ; 10(1): 11880, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681023

RESUMO

Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot (Scophthalmus maximus). In addition to a cytochrome pathway (CP), the ciliate can use a cyanide-insensitive respiratory pathway, which indicates the existence of an alternative oxidase (AOX) in the mitochondrion. Although AOX activity has been described in P. dicentrarchi, based on functional assay results, genetic evidence of the presence of AOX in the ciliate has not previously been reported. In this study, we conducted genomic and transcriptomic analysis of the ciliate and identified the AOX gene and its corresponding mRNA. The AOX gene (size 1,106 bp) contains four exons and three introns that generate an open reading frame of 915 bp and a protein with a predicted molecular weight of 35.6 kDa. The amino acid (aa) sequence of the AOX includes an import signal peptide targeting the mitochondria and the protein is associated with the inner membrane of the mitochondria. Bioinformatic analysis predicted that the peptide is a homodimeric glycoprotein, although monomeric forms may also appear under native conditions, with EXXH motifs associated with the diiron active centers. The aa sequences of the AOX of different P. dicentrarchi isolates are highly conserved and phylogenetically closely related to AOXs of other ciliate species, especially scuticociliates. AOX expression increased significantly during infection in the host and after the addition of CP inhibitors. This confirms the important physiological roles of AOX in respiration under conditions of low levels of O2 and in protecting against oxidative stress generated during infection in the host.


Assuntos
Respiração Celular/efeitos dos fármacos , Hipóxia/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Oligoimenóforos/enzimologia , Oligoimenóforos/genética , Oxirredutases/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Expressão Gênica , Genoma de Protozoário , Genômica/métodos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/química , Modelos Moleculares , Oligoimenóforos/classificação , Oxirredutases/química , Filogenia , Proteínas de Plantas/química , Conformação Proteica , Relação Estrutura-Atividade
17.
PLoS Genet ; 16(7): e1008949, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32702045

RESUMO

In Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and DNA excision and repair. How germline limited DNA sequences are specifically recognized in the context of chromatin remains elusive. Here, we use a reverse genetics approach to identify factors involved in programmed genome rearrangements. We have identified a P. tetraurelia homolog of the highly conserved histone chaperone Spt16 subunit of the FACT complex, Spt16-1, and show its expression is developmentally regulated. A functional GFP-Spt16-1 fusion protein localized exclusively in the nuclei where genome rearrangements take place. Gene silencing of Spt16-1 showed it is required for the elimination of all germline-limited sequences, for the survival of sexual progeny, and for the accumulation of internal eliminated sequence (ies)RNAs, an sRNA population produced when elimination occurs. Normal accumulation of 25 nt scanRNAs and deposition of silent histone marks H3K9me3 and H3K27me3 indicated that Spt16-1 does not regulate the scanRNA-directed heterochromatin pathway involved in the early steps of DNA elimination. We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination. Thus, Spt16-1 is essential for Pgm function during programmed genome rearrangements. We propose a model in which Spt16-1 mediates interactions between the excision machinery and chromatin, facilitating endonuclease access to DNA cleavage sites during genome rearrangements.


Assuntos
Núcleo Celular/genética , Chaperonas de Histonas/genética , Paramecium/genética , Transposases/genética , Sequência de Bases/genética , Quebras de DNA de Cadeia Dupla , Clivagem do DNA , DNA de Protozoário/genética , Endonucleases , Rearranjo Gênico/genética , Genoma de Protozoário/genética , Paramecium/crescimento & desenvolvimento
18.
Sci Rep ; 10(1): 9792, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32555361

RESUMO

Dinoflagellates of the Symbiodiniaceae family encompass diverse symbionts that are critical to corals and other species living in coral reefs. It is well known that sexual reproduction enhances adaptive evolution in changing environments. Although genes related to meiotic functions were reported in Symbiodiniaceae, cytological evidence of meiosis and fertilisation are however yet to be observed in these taxa. Using transcriptome and genome data from 21 Symbiodiniaceae isolates, we studied genes that encode proteins associated with distinct stages of meiosis and syngamy. We report the absence of genes that encode main components of the synaptonemal complex (SC), a protein structure that mediates homologous chromosomal pairing and class I crossovers. This result suggests an independent loss of canonical SCs in the alveolates, that also includes the SC-lacking ciliates. We hypothesise that this loss was due in part to permanently condensed chromosomes and repeat-rich sequences in Symbiodiniaceae (and other dinoflagellates) which favoured the SC-independent class II crossover pathway. Our results reveal novel insights into evolution of the meiotic molecular machinery in the ecologically important Symbiodiniaceae and in other eukaryotes.


Assuntos
Evolução Biológica , Dinoflagelados/fisiologia , Complexo Sinaptonêmico/genética , Uso do Códon , Conjuntos de Dados como Assunto , Dinoflagelados/genética , Perfilação da Expressão Gênica , Genoma de Protozoário , Meiose/genética , Seleção Genética , Sexo
19.
Sci Rep ; 10(1): 9279, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518251

RESUMO

Honey bees are large-scale monitoring tools due to their extensive environmental exploration. In their activities and from the hive ecosystem complex, they get in close contact with many organisms whose traces can be transferred into the honey, which can represent an interesting reservoir of environmental DNA (eDNA) signatures and information useful to analyse the honey bee hologenome complexity. In this study, we tested a deep shotgun sequencing approach of honey DNA coupled with a specifically adapted bioinformatic pipeline. This methodology was applied to a few honey samples pointing out DNA sequences from 191 organisms spanning different kingdoms or phyla (viruses, bacteria, plants, fungi, protozoans, arthropods, mammals). Bacteria included the largest number of species. These multi-kingdom signatures listed common hive and honey bee gut microorganisms, honey bee pathogens, parasites and pests, which resembled a complex interplay that might provide a general picture of the honey bee pathosphere. Based on the Apis mellifera filamentous virus genome diversity (the most abundant detected DNA source) we obtained information that could define the origin of the honey at the apiary level. Mining Apis mellifera sequences made it possible to identify the honey bee subspecies both at the mitochondrial and nuclear genome levels.


Assuntos
DNA Ambiental/análise , Mel/análise , Metagenômica , Animais , Bactérias/genética , Abelhas , DNA Ambiental/genética , Microbioma Gastrointestinal/genética , Genoma Bacteriano/genética , Genoma Fúngico/genética , Genoma de Planta/genética , Genoma de Protozoário/genética , Genoma Viral/genética , Análise de Sequência de DNA
20.
Sci Rep ; 10(1): 7316, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355272

RESUMO

Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.


Assuntos
Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , Fatores de Virulência/genética , Virulência/genética , Animais , Sistemas CRISPR-Cas , Carcinogênese/genética , Biologia Computacional , Cryptosporidium parvum/patogenicidade , Fezes , Feminino , Genoma , Genoma de Protozoário , Humanos , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Oocistos , Fenótipo , Adulto Jovem
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