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2.
PLoS One ; 17(9): e0270863, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36048819

RESUMO

Plasmodium falciparum, a protozoan parasite and causative agent of human malaria, has one of the most A/T-biased genomes sequenced to date. This may give the genome and the transcriptome unusual structural features. Recent progress in sequencing techniques has made it possible to study the secondary structures of RNA molecules at the transcriptomic level. Thus, in this study we produced the in vivo RNA structurome of a protozoan parasite with a highly A/U-biased transcriptome. We showed that it is possible to probe the secondary structures of P. falciparum RNA molecules in vivo using two different chemical probes, and obtained structures for more than half of all transcripts in the transcriptome. These showed greater stability (lower free energy) than the same structures modelled in silico, and structural features appeared to influence translation efficiency and RNA decay. Finally, we compared the P. falciparum RNA structurome with the predicted RNA structurome of an A/U-balanced species, P. knowlesi, finding a bias towards lower overall transcript stability and more hairpins and multi-stem loops in P. falciparum. This unusual protozoan RNA structurome will provide a basis for similar studies in other protozoans and also in other unusual genomes.


Assuntos
Malária Falciparum , Malária , Parasitos , Animais , Genoma de Protozoário , Humanos , Malária/genética , Malária Falciparum/parasitologia , Parasitos/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , RNA , RNA de Protozoário/genética , Transcriptoma
3.
Sci Data ; 9(1): 585, 2022 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-36153341

RESUMO

Spironucleus salmonicida is a diplomonad causing systemic infection in salmon. The first S. salmonicida genome assembly was published 2014 and has been a valuable reference genome in protist research. However, the genome assembly is fragmented without assignment of the sequences to chromosomes. In our previous Giardia genome study, we have shown how a fragmented genome assembly can be improved with long-read sequencing technology complemented with optical maps. Combining Pacbio long-read sequencing technology and optical maps, we are presenting here this new S. salmonicida genome assembly in nine near-complete chromosomes with only three internal gaps at long repeats. This new genome assembly is not only more complete sequence-wise but also more complete at annotation level, providing more details into gene families, gene organizations and chromosomal structure. This near-complete reference genome will aid comparative genomics at chromosomal level, and serve as a valuable resource for the diplomonad community and protist research.


Assuntos
Diplomonadida , Genoma de Protozoário , Cromossomos/genética , Diplomonadida/genética , Genômica , Anotação de Sequência Molecular , Análise de Sequência de DNA
4.
Methods Mol Biol ; 2470: 221-239, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35881349

RESUMO

The ability to interrogate gene function in Plasmodium parasites has been greatly enhanced by the advent of CRISPR/Cas9 systems. The breadth of genome manipulations ranges from single point mutations to large multigene deletions, however many of the technical considerations for designing CRISPR-based experiments are common to any editing approach. This review will discuss protocols for vector construction and donor design for genome editing P. falciparum, including pitfalls, variables, and validation methods.


Assuntos
Plasmodium falciparum , Plasmodium , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma de Protozoário , Plasmodium falciparum/genética
5.
J Eukaryot Microbiol ; 69(5): e12914, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35363910

RESUMO

The term epigenetics is used for any layer of genetic information aside from the DNA base-sequence information. Mammalian epigenetic research increased our understanding of chromatin dynamics in terms of cytosine methylation and histone modification during differentiation, aging, and disease. Instead, ciliate epigenetics focused more on small RNA-mediated effects. On the one hand, these do concern the transport of RNA from parental to daughter nuclei, representing a regulated transfer of epigenetic information across generations. On the other hand, studies of Paramecium, Tetrahymena, Oxytricha, and Stylonychia revealed an almost unique function of transgenerational RNA. Rather than solely controlling chromatin dynamics, they control sexual progeny's DNA content quantitatively and qualitatively. Thus epigenetics seems to control genetics, at least genetics of the vegetative macronucleus. This combination offers ciliates, in particular, an epigenetically controlled genetic variability. This review summarizes the epigenetic mechanisms that contribute to macronuclear heterogeneity and relates these to nuclear dimorphism. This system's adaptive and evolutionary possibilities raise the critical question of whether such a system is limited to unicellular organisms or binuclear cells. We discuss here the relevance of ciliate genetics and epigenetics to multicellular organisms.


Assuntos
Cilióforos , Paramecium , Animais , Proliferação de Células , Cromatina , Cilióforos/genética , DNA , Epigênese Genética , Genoma de Protozoário , Mamíferos , Paramecium/genética , RNA
6.
Front Cell Infect Microbiol ; 12: 760830, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35402315

RESUMO

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits extensive inter- and intrastrain genetic diversity. As we have previously described, there are some genetic differences between the parental G strain and its clone D11, which was isolated by the limiting dilution method and infection of cultured mammalian cells. Electrophoretic karyotyping and Southern blot hybridization of chromosomal bands with specific markers revealed chromosome length polymorphisms of small size with additional chromosomal bands in clone D11 and the maintenance of large syntenic groups. Both G strain and clone D11 belong to the T. cruzi lineage TcI. Here, we designed intraspecific array-based comparative genomic hybridization (aCGH) to identify chromosomal regions harboring copy-number variations between clone D11 and the G strain. DNA losses were more extensive than DNA gains in clone D11. Most alterations were flanked by repeated sequences from multigene families that could be involved in the duplication and deletion events. Several rearrangements were detected by chromoblot hybridization and confirmed by aCGH. We have integrated the information of genomic sequence data obtained by aCGH to the electrophoretic karyotype, allowing the reconstruction of possible recombination events that could have generated the karyotype of clone D11. These rearrangements may be explained by unequal crossing over between sister or homologous chromatids mediated by flanking repeated sequences and unequal homologous recombination via break-induced replication. The genomic changes detected by aCGH suggest the presence of a dynamic genome that responds to environmental stress by varying the number of gene copies and generating segmental aneuploidy.


Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Células Clonais , Hibridização Genômica Comparativa/métodos , DNA , Genoma de Protozoário , Mamíferos/genética , Trypanosoma cruzi/genética
7.
Mol Biol Evol ; 39(4)2022 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-35325184

RESUMO

Ciliated protists are among the oldest unicellular organisms with a heterotrophic lifestyle and share a common ancestor with Plantae. Unlike any other eukaryotes, there are two distinct nuclei in ciliates with separate germline and somatic cell functions. Here, we assembled a near-complete macronuclear genome of Fabrea salina, which belongs to one of the oldest clades of ciliates. Its extremely minimized genome (18.35 Mb) is the smallest among all free-living heterotrophic eukaryotes and exhibits typical streamlined genomic features, including high gene density, tiny introns, and shrinkage of gene paralogs. Gene families involved in hypersaline stress resistance, DNA replication proteins, and mitochondrial biogenesis are expanded, and the accumulation of phosphatidic acid may play an important role in resistance to high osmotic pressure. We further investigated the morphological and transcriptomic changes in the macronucleus during sexual reproduction and highlighted the potential contribution of macronuclear residuals to this process. We believe that the minimized genome generated in this study provides novel insights into the genome streamlining theory and will be an ideal model to study the evolution of eukaryotic heterotrophs.


Assuntos
Cilióforos , Genoma de Protozoário , Cilióforos/genética , DNA de Protozoário/genética , Íntrons , Macronúcleo/genética , Análise de Sequência de DNA
8.
PLoS Comput Biol ; 18(2): e1009870, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35196325

RESUMO

Protozoan parasites cause diverse diseases with large global impacts. Research on the pathogenesis and biology of these organisms is limited by economic and experimental constraints. Accordingly, studies of one parasite are frequently extrapolated to infer knowledge about another parasite, across and within genera. Model in vitro or in vivo systems are frequently used to enhance experimental manipulability, but these systems generally use species related to, yet distinct from, the clinically relevant causal pathogen. Characterization of functional differences among parasite species is confined to post hoc or single target studies, limiting the utility of this extrapolation approach. To address this challenge and to accelerate parasitology research broadly, we present a functional comparative analysis of 192 genomes, representing every high-quality, publicly-available protozoan parasite genome including Plasmodium, Toxoplasma, Cryptosporidium, Entamoeba, Trypanosoma, Leishmania, Giardia, and other species. We generated an automated metabolic network reconstruction pipeline optimized for eukaryotic organisms. These metabolic network reconstructions serve as biochemical knowledgebases for each parasite, enabling qualitative and quantitative comparisons of metabolic behavior across parasites. We identified putative differences in gene essentiality and pathway utilization to facilitate the comparison of experimental findings and discovered that phylogeny is not the sole predictor of metabolic similarity. This knowledgebase represents the largest collection of genome-scale metabolic models for both pathogens and eukaryotes; with this resource, we can predict species-specific functions, contextualize experimental results, and optimize selection of experimental systems for fastidious species.


Assuntos
Criptosporidiose , Cryptosporidium , Parasitos , Plasmodium , Animais , Criptosporidiose/genética , Cryptosporidium/genética , Eucariotos/genética , Genoma de Protozoário/genética , Parasitos/genética , Plasmodium/genética
9.
Nucleic Acids Res ; 50(5): 2603-2620, 2022 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-35188560

RESUMO

The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline and somatic nuclei, massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs - 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.


Assuntos
Genoma de Protozoário , Paramecium tetraurellia , Meiose , Paramecium tetraurellia/citologia , Paramecium tetraurellia/genética , Paramecium tetraurellia/crescimento & desenvolvimento , RNA não Traduzido/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
10.
PLoS Pathog ; 18(2): e1010288, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35167626

RESUMO

Urogenital schistosomiasis is caused by the blood fluke Schistosoma haematobium and is one of the most neglected tropical diseases worldwide, afflicting > 100 million people. It is characterised by granulomata, fibrosis and calcification in urogenital tissues, and can lead to increased susceptibility to HIV/AIDS and squamous cell carcinoma of the bladder. To complement available treatment programs and break the transmission of disease, sound knowledge and understanding of the biology and ecology of S. haematobium is required. Hybridisation/introgression events and molecular variation among members of the S. haematobium-group might effect important biological and/or disease traits as well as the morbidity of disease and the effectiveness of control programs including mass drug administration. Here we report the first chromosome-contiguous genome for a well-defined laboratory line of this blood fluke. An exploration of this genome using transcriptomic data for all key developmental stages allowed us to refine gene models (including non-coding elements) and annotations, discover 'new' genes and transcription profiles for these stages, likely linked to development and/or pathogenesis. Molecular variation within S. haematobium among some geographical locations in Africa revealed unique genomic 'signatures' that matched species other than S. haematobium, indicating the occurrence of introgression events. The present reference genome (designated Shae.V3) and the findings from this study solidly underpin future functional genomic and molecular investigations of S. haematobium and accelerate systematic, large-scale population genomics investigations, with a focus on improved and sustained control of urogenital schistosomiasis.


Assuntos
Variação Genética , Genoma de Protozoário , Schistosoma haematobium/genética , Esquistossomose Urinária/parasitologia , Transcriptoma , Animais , Cromossomos/parasitologia , Genes de Protozoários , Genoma , Estudo de Associação Genômica Ampla , Análise de Sequência de DNA
11.
Mol Genet Genomics ; 297(1): 1-18, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34999963

RESUMO

Genome sequence analysis of Entamoeba species revealed various classes of transposable elements. While E. histolytica and E. dispar are rich in non-long terminal repeat (LTR) retrotransposons, E. invadens contains predominantly DNA transposons. Non-LTR retrotransposons of E. histolytica constitute three families of long interspersed nuclear elements (LINEs), and their short, nonautonomous partners, SINEs. They occupy ~ 11% of the genome. The EhLINE1/EhSINE1 family is the most abundant and best studied. EhLINE1 is 4.8 kb, with two ORFs that encode functions needed for retrotransposition. ORF1 codes for the nucleic acid-binding protein, and ORF2 has domains for reverse transcriptase (RT) and endonuclease (EN). Most copies of EhLINEs lack complete ORFs. ORF1p is expressed constitutively, but ORF2p is not detected. Retrotransposition could be demonstrated upon ectopic over expression of ORF2p, showing that retrotransposition machinery is functional. The newly retrotransposed sequences showed a high degree of recombination. In transcriptomic analysis, RNA-Seq reads were mapped to individual EhLINE1 copies. Although full-length copies were transcribed, no full-length 4.8 kb transcripts were seen. Rather, sense transcripts mapped to ORF1, RT and EN domains. Intriguingly, there was strong antisense transcription almost exclusively from the RT domain. These unique features of EhLINE1 could serve to attenuate retrotransposition in E. histolytica.


Assuntos
Entamoeba histolytica/genética , Entamoeba histolytica/fisiologia , Animais , Mapeamento Cromossômico , Genoma de Protozoário/genética , Genômica , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Fases de Leitura Aberta/genética , Retroelementos , Elementos Nucleotídeos Curtos e Dispersos/genética
12.
Nucleic Acids Res ; 50(1): 293-305, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-34893872

RESUMO

Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.


Assuntos
Aneuploidia , Evolução Molecular , Leishmania donovani/genética , Mosaicismo , Análise de Célula Única/métodos , Sequenciamento Completo do Genoma/métodos , Genoma de Protozoário
13.
PLoS Negl Trop Dis ; 15(9): e0009504, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34543277

RESUMO

Trypanosoma brucei ssp., unicellular parasites causing human and animal trypanosomiasis, are transmitted between mammals by tsetse flies. Periodic changes in variant surface glycoproteins (VSG), which form the parasite coat in the mammal, allow them to evade the host immune response. Different isolates of T. brucei show heterogeneity in their repertoires of VSG genes and have single nucleotide polymorphisms and indels that can impact on genome editing. T. brucei brucei EATRO1125 (AnTaR1 serodeme) is an isolate that is used increasingly often because it is pleomorphic in mammals and fly transmissible, two characteristics that have been lost by the most commonly used laboratory stocks. We present a genome assembly of EATRO1125, including contigs for the intermediate chromosomes and minichromosomes that serve as repositories of VSG genes. In addition, de novo transcriptome assemblies were performed using Illumina sequences from tsetse-derived trypanosomes. Reads of 150 bases enabled closely related members of multigene families to be discriminated. This revealed that the transcriptome of midgut-derived parasites is dynamic, starting with the expression of high affinity hexose transporters and glycolytic enzymes and then switching to proline uptake and catabolism. These changes resemble the transition from early to late procyclic forms in culture. Further metabolic reprogramming, including upregulation of tricarboxylic acid cycle enzymes, occurs in the proventriculus. Many transcripts upregulated in the salivary glands encode surface proteins, among them 7 metacyclic VSGs, multiple BARPs and GCS1/HAP2, a marker for gametes. A novel family of transmembrane proteins, containing polythreonine stretches that are predicted to be O-glycosylation sites, was also identified. Finally, RNA-Seq data were used to create an optimised annotation file with 5' and 3' untranslated regions accurately mapped for 9302 genes. We anticipate that this will be of use in identifying transcripts obtained by single cell sequencing technologies.


Assuntos
DNA de Protozoário/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Insetos Vetores/parasitologia , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/fisiologia , Moscas Tsé-Tsé/parasitologia , Animais , Metabolismo Energético , Perfilação da Expressão Gênica , Genoma de Protozoário , Interações Hospedeiro-Parasita , Proteínas de Protozoários/genética , RNA-Seq , Glândulas Salivares/parasitologia
14.
Microb Genom ; 7(12)2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34907893

RESUMO

Cryptosporidium spp. are important enteric pathogens in a wide range of vertebrates including humans. Previous comparative analysis revealed conservation in genome composition, gene content, and gene organization among Cryptosporidium spp., with a progressive reductive evolution in metabolic pathways and invasion-related proteins. In this study, we sequenced the genome of zoonotic pathogen Cryptosporidium felis and conducted a comparative genomic analysis. While most intestinal Cryptosporidium species have similar genomic characteristics and almost complete genome synteny, fewer protein-coding genes and some sequence inversions and translocations were found in the C. felis genome. The C. felis genome exhibits much higher GC content (39.6 %) than other Cryptosporidium species (24.3-32.9 %), especially at the third codon position (GC3) of protein-coding genes. Thus, C. felis has a different codon usage, which increases the use of less energy costly amino acids (Gly and Ala) encoded by GC-rich codons. While the tRNA usage is conserved among Cryptosporidium species, consistent with its higher GC content, C. felis uses a unique tRNA for GTG for valine instead of GTA in other Cryptosporidium species. Both mutational pressures and natural selection are associated with the evolution of the codon usage in Cryptosporidium spp., while natural selection seems to drive the codon usage in C. felis. Other unique features of the C. felis genome include the loss of the entire traditional and alternative electron transport systems and several invasion-related proteins. Thus, the preference for the use of some less energy costly amino acids in C. felis may lead to a more harmonious parasite-host interaction, and the strengthened host-adaptation is reflected by the further reductive evolution of metabolism and host invasion-related proteins.


Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/classificação , Genoma de Protozoário , Sequenciamento Completo do Genoma/métodos , Composição de Bases , Uso do Códon , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Filogenia , Seleção Genética , Sintenia
15.
Int J Mol Sci ; 22(23)2021 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-34884870

RESUMO

The parasite species of genus Plasmodium causes Malaria, which remains a major global health problem due to parasite resistance to available Antimalarial drugs and increasing treatment costs. Consequently, computational prediction of new Antimalarial compounds with novel targets in the proteome of Plasmodium sp. is a very important goal for the pharmaceutical industry. We can expect that the success of the pre-clinical assay depends on the conditions of assay per se, the chemical structure of the drug, the structure of the target protein to be targeted, as well as on factors governing the expression of this protein in the proteome such as genes (Deoxyribonucleic acid, DNA) sequence and/or chromosomes structure. However, there are no reports of computational models that consider all these factors simultaneously. Some of the difficulties for this kind of analysis are the dispersion of data in different datasets, the high heterogeneity of data, etc. In this work, we analyzed three databases ChEMBL (Chemical database of the European Molecular Biology Laboratory), UniProt (Universal Protein Resource), and NCBI-GDV (National Center for Biotechnology Information-Genome Data Viewer) to achieve this goal. The ChEMBL dataset contains outcomes for 17,758 unique assays of potential Antimalarial compounds including numeric descriptors (variables) for the structure of compounds as well as a huge amount of information about the conditions of assays. The NCBI-GDV and UniProt datasets include the sequence of genes, proteins, and their functions. In addition, we also created two partitions (cassayj = caj and cdataj = cdj) of categorical variables from theChEMBL dataset. These partitions contain variables that encode information about experimental conditions of preclinical assays (caj) or about the nature and quality of data (cdj). These categorical variables include information about 22 parameters of biological activity (ca0), 28 target proteins (ca1), and 9 organisms of assay (ca2), etc. We also created another partition of (cprotj = cpj) including categorical variables with biological information about the target proteins, genes, and chromosomes. These variables cover32 genes (cp0), 10 chromosomes (cp1), gene orientation (cp2), and 31 protein functions (cp3). We used a Perturbation-Theory Machine Learning Information Fusion (IFPTML) algorithm to map all this information (from three databases) into and train a predictive model. Shannon's entropy measure Shk (numerical variables) was used to quantify the information about the structure of drugs, protein sequences, gene sequences, and chromosomes in the same information scale. Perturbation Theory Operators (PTOs) with the form of Moving Average (MA) operators have been used to quantify perturbations (deviations) in the structural variables with respect to their expected values for different subsets (partitions) of categorical variables. We obtained three IFPTML models using General Discriminant Analysis (GDA), Classification Tree with Univariate Splits (CTUS), and Classification Tree with Linear Combinations (CTLC). The IFPTML-CTLC presented the better performance with Sensitivity Sn(%) = 83.6/85.1, and Specificity Sp(%) = 89.8/89.7 for training/validation sets, respectively. This model could become a useful tool for the optimization of preclinical assays of new Antimalarial compounds vs. different proteins in the proteome of Plasmodium.


Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas/métodos , Aprendizado de Máquina , Plasmodium falciparum/genética , Algoritmos , Antimaláricos/química , Bases de Dados de Produtos Farmacêuticos , Avaliação Pré-Clínica de Medicamentos , Genoma de Protozoário , Cadeias de Markov , Modelos Teóricos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Reprodutibilidade dos Testes
16.
PLoS Negl Trop Dis ; 15(12): e0010041, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34965251

RESUMO

BACKGROUND: Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. CONCLUSION/SIGNIFICANCE: Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.


Assuntos
Instabilidade Genômica , Autoantígeno Ku/metabolismo , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Proteínas de Protozoários/metabolismo , Telômero/metabolismo , Genoma de Protozoário , Humanos , Autoantígeno Ku/genética , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/genética , Telômero/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo
17.
PLoS Negl Trop Dis ; 15(12): e0010110, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34968388

RESUMO

BACKGROUND: The kinetoplastid protozoan Leishmania tropica mainly causes cutaneous leishmaniasis in humans in the Middle East, and relapse or treatment failure after treatment are common in this area. L. tropica's digenic life cycle includes distinct stages in the vector sandfly and the mammalian host. Sexual reproduction and genetic exchange appear to occur more frequently than in other Leishmania species. Understanding these processes is complicated by chromosome instability during cell division that yields aneuploidy, recombination and heterozygosity. This combination of rare recombination and aneuploid permits may reveal signs of hypothetical parasexual mating, where diploid cells fuse to form a transient tetraploid that undergoes chromosomal recombination and gradual chromosomal loss. METHODOLOGY/PRINCIPAL FINDINGS: The genome-wide SNP diversity from 22 L. tropica isolates showed chromosome-specific runs of patchy heterozygosity and extensive chromosome copy number variation. All these isolates were collected during 2007-2017 in Sweden from patients infected in the Middle East and included isolates from a patient possessing two genetically distinct leishmaniasis infections three years apart with no evidence of re-infection. We found differing ancestries on the same chromosome (chr36) across multiple samples: matching the reference genome with few derived alleles, followed by blocks of heterozygous SNPs, and then by clusters of homozygous SNPs with specific recombination breakpoints at an inferred origin of replication. Other chromosomes had similar marked changes in heterozygosity at strand-switch regions separating polycistronic transcriptional units. CONCLUSION/SIGNIFICANCE: These large-scale intra- and inter-chromosomal changes in diversity driven by recombination and aneuploidy suggest multiple mechanisms of cell reproduction and diversification in L. tropica, including mitotic, meiotic and parasexual processes. It underpins the need for more genomic surveillance of Leishmania, to detect emerging hybrids that could spread more widely and to better understand the association between genetic variation and treatment outcome. Furthering our understanding of Leishmania genome evolution and ancestry will aid better diagnostics and treatment for cutaneous leishmaniasis caused by L.tropica in the Middle East.


Assuntos
Genoma de Protozoário , Leishmania tropica/genética , Leishmaniose Cutânea/parasitologia , Afeganistão , Cromossomos/genética , Variações do Número de Cópias de DNA , DNA de Protozoário/genética , Variação Genética , Humanos , Irã (Geográfico) , Leishmania tropica/classificação , Leishmania tropica/isolamento & purificação , Filogenia , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Síria
18.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34903666

RESUMO

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2'-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.


Assuntos
Adaptação Fisiológica/genética , Epistasia Genética , Genoma de Protozoário , Instabilidade Genômica , Leishmania/genética , Dosagem de Genes , Aptidão Genética , Humanos , Leishmaniose/parasitologia
19.
mBio ; 12(6): e0135221, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34749530

RESUMO

The parasite Trypanosoma brucei periodically changes the expression of protective variant surface glycoproteins (VSGs) to evade its host's immune system in a process known as antigenic variation. One route to change VSG expression is the transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machinery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We identified several novel DOT1B interactors. One of these was the RNase H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage, and ES switching events. Surprisingly, a similar pattern of VSG deregulation was observed in RNase H2 mutants. We propose that both proteins act together in resolving R-loops to ensure genome integrity and contribute to the tightly regulated process of antigenic variation. IMPORTANCE Trypanosoma brucei is a unicellular parasite that causes devastating diseases like sleeping sickness in humans and the "nagana" disease in cattle in Africa. Fundamental to the establishment and prolongation of a trypanosome infection is the parasite's ability to escape the mammalian host's immune system by antigenic variation, which relies on periodic changes of a protein surface coat. The exact mechanisms, however, which mediate these changes are still elusive. In this work, we describe a novel protein complex consisting of the histone methyltransferase DOT1B and RNase H2 which is involved in antigenic variation.


Assuntos
Histona Metiltransferases/metabolismo , Proteínas de Protozoários/metabolismo , Ribonuclease H/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanossomíase/parasitologia , Variação Antigênica , Genoma de Protozoário , Instabilidade Genômica , Histona Metiltransferases/química , Histona Metiltransferases/genética , Humanos , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Estruturas R-Loop , Ribonuclease H/química , Ribonuclease H/genética , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/enzimologia
20.
STAR Protoc ; 2(4): 100936, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34806047

RESUMO

This protocol describes the genomic phage (gPhage) display platform, a large-scale antigen and epitope mapping technique. We constructed a gPhage display peptide library of a eukaryotic organism, Trypanosoma cruzi (causative agent of Chagas disease), to map the antibody response landscape against the parasite. Here, we used an organism with a relatively large but intronless genome, although future applications could include other prevalent or (re)emerging infectious organisms carrying genomes with a limited number of introns. For complete details on the use and execution of this protocol, please refer to Teixeira et al. (2021).


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Biblioteca Genômica , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/metabolismo , Genoma de Protozoário/genética , Trypanosoma cruzi/genética
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