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1.
Mem Inst Oswaldo Cruz ; 116: e200634, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33787768

RESUMO

The availability of Trypanosomatid genomic data in public databases has opened myriad experimental possibilities that have contributed to a more comprehensive understanding of the biology of these parasites and their interactions with hosts. In this review, after brief remarks on the history of the Trypanosoma cruzi and Leishmania genome initiatives, we present an overview of the relevant contributions of genomics, transcriptomics and functional genomics, discussing the primary obstacles, challenges, relevant achievements and future perspectives of these technologies.


Assuntos
Genoma de Protozoário/genética , Leishmania/genética , Trypanosoma cruzi/genética , Biologia Computacional , Genômica
2.
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
3.
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
4.
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
5.
Cell Mol Life Sci ; 77(22): 4615-4629, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32462406

RESUMO

Ciliates are a highly divergent group of unicellular eukaryotes with separate somatic and germline genomes found in distinct dimorphic nuclei. This characteristic feature is tightly linked to extremely laborious developmentally regulated genome rearrangements in the development of a new somatic genome/nuclei following sex. The transformation from germline to soma genome involves massive DNA elimination mediated by non-coding RNAs, chromosome fragmentation, as well as DNA amplification. In this review, we discuss the similarities and differences in the genome reorganization processes of the model ciliates Paramecium and Tetrahymena (class Oligohymenophorea), and the distantly related Euplotes, Stylonychia, and Oxytricha (class Spirotrichea).


Assuntos
Cilióforos/genética , Rearranjo Gênico/genética , Genoma de Protozoário/genética , Animais , Núcleo Celular/genética , Células Germinativas/fisiologia , Humanos
6.
Parasitol Res ; 119(4): 1401-1408, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32130486

RESUMO

Fatty acid-binding proteins (FABPs) are small intracellular proteins that reversibly bind fatty acids and other hydrophobic ligands. In cestodes, due to their inability to synthesise fatty acids de novo, FABPs have been proposed as essential proteins, and thus, as possible drug targets and/or carriers against these parasites. We performed data mining in Echinococcus multilocularis and Echinococcus granulosus genomes in order to test whether this family of proteins is more complex than previously reported. By exploring the genomes of E. multilocularis and E. granulosus, six genes coding for FABPs were found in each organism. In the case of E. granulosus, all of them have different coding sequences, whereas in E. multilocularis, two of the genes code for the same protein. Remarkably, one of the genes (in both cestodes) encodes a FABP with a C-terminal extension unusual for this family of proteins. The newly described genes present variations in their structure in comparison with previously described FABP genes in Echinococcus spp. The coding sequences for E. multilocularis were validated by cloning and sequencing. Moreover, differential expression patterns of FABPs were observed at different stages of the life cycle of E. multilocularis by exploring transcriptomic data from several sources. In summary, FABP family in cestodes is far more complex than previously thought and includes new members that seem to be only present in flatworms.


Assuntos
Echinococcus granulosus/genética , Echinococcus multilocularis/genética , Proteínas de Ligação a Ácido Graxo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA de Protozoário/genética , Ácidos Graxos/metabolismo , Genoma de Protozoário/genética , Análise de Sequência , Análise de Sequência de DNA , Transcriptoma/genética
7.
Trends Parasitol ; 36(4): 318-321, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32191848

RESUMO

The mitochondrion in parasitic protozoans is a clinically proven drug target. A specialized ribosome (mitoribosome) is required to translate genes encoded on the mitochondrial (mt) DNA. Despite the significance, little is known about mitoribosomes in many medically and economically important unicellular protozoans.


Assuntos
Eucariotos/genética , Variação Genética , Genoma de Protozoário/genética , Ribossomos Mitocondriais , Parasitos/genética , Animais , Genoma Mitocondrial/genética
8.
Trends Parasitol ; 36(4): 356-367, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32191850

RESUMO

Elimination programs targeting TriTryp diseases (Leishmaniasis, Chagas' disease, human African trypanosomiasis) significantly reduced the number of cases. Continued surveillance is crucial to sustain this progress, but parasite molecular surveillance by genotyping is currently lacking. We explain here which epidemiological questions of public health and clinical relevance could be answered by means of molecular surveillance. Whole-genome sequencing (WGS) for molecular surveillance will be an important added value, where we advocate that preference should be given to direct sequencing of the parasite's genome in host tissues instead of analysis of cultivated isolates. The main challenges here, and recent technological advances, are discussed. We conclude with a series of recommendations for implementing whole-genome sequencing for molecular surveillance.


Assuntos
Infecções por Euglenozoa/prevenção & controle , Infecções por Euglenozoa/parasitologia , Genoma de Protozoário/genética , Biologia Molecular/tendências , Infecções por Euglenozoa/epidemiologia , Humanos , Pesquisa/tendências , Sequenciamento Completo do Genoma/tendências
9.
PLoS Genet ; 16(2): e1008576, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32053607

RESUMO

Although Plasmodium vivax parasites are the predominant cause of malaria outside of sub-Saharan Africa, they not always prioritised by elimination programmes. P. vivax is resilient and poses challenges through its ability to re-emerge from dormancy in the human liver. With observed growing drug-resistance and the increasing reports of life-threatening infections, new tools to inform elimination efforts are needed. In order to halt transmission, we need to better understand the dynamics of transmission, the movement of parasites, and the reservoirs of infection in order to design targeted interventions. The use of molecular genetics and epidemiology for tracking and studying malaria parasite populations has been applied successfully in P. falciparum species and here we sought to develop a molecular genetic tool for P. vivax. By assembling the largest set of P. vivax whole genome sequences (n = 433) spanning 17 countries, and applying a machine learning approach, we created a 71 SNP barcode with high predictive ability to identify geographic origin (91.4%). Further, due to the inclusion of markers for within population variability, the barcode may also distinguish local transmission networks. By using P. vivax data from a low-transmission setting in Malaysia, we demonstrate the potential ability to infer outbreak events. By characterising the barcoding SNP genotypes in P. vivax DNA sourced from UK travellers (n = 132) to ten malaria endemic countries predominantly not used in the barcode construction, we correctly predicted the geographic region of infection origin. Overall, the 71 SNP barcode outperforms previously published genotyping methods and when rolled-out within new portable platforms, is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria.


Assuntos
Surtos de Doenças/prevenção & controle , Genoma de Protozoário/genética , Técnicas de Genotipagem/métodos , Malária Vivax/transmissão , Plasmodium vivax/genética , África Oriental , Ásia , Conjuntos de Dados como Assunto , Erradicação de Doenças/métodos , Marcadores Genéticos/genética , Genótipo , Geografia , Humanos , Malária Vivax/epidemiologia , Malária Vivax/parasitologia , Metadados , Repetições de Microssatélites/genética , Plasmodium vivax/isolamento & purificação , Polimorfismo de Nucleotídeo Único/genética , Valor Preditivo dos Testes , América do Sul , Doença Relacionada a Viagens , Reino Unido , Sequenciamento Completo do Genoma
10.
Trends Parasitol ; 36(2): 83-85, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31883706

RESUMO

Recent genomic studies are investigating the wide-ranging implications of malaria complex infections on parasite diversity, transmission, and downstream disease eradication efforts. Using single cell sequencing, new work by Nkhoma et al. provides evidence that there is unexpectedly frequent co-transmission of related parasites in the intense transmission setting in Malawi.


Assuntos
Malária/parasitologia , Plasmodium/classificação , Plasmodium/genética , Animais , Genoma de Protozoário/genética , Genômica , Interações Hospedeiro-Parasita , Humanos , Malária/transmissão , Filogenia , Análise de Célula Única
11.
Parasitology ; 147(1): 29-38, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31452478

RESUMO

The presence of bacterial DNA in Dientamoeba fragilis DNA extracts from culture poses a substantial challenge to sequencing the D. fragilis genome. However, elimination of bacteria from D. fragilis cultures has proven difficult in the past, presumably due to its dependence on some unknown prokaryote/s. This study explored options for removal of bacteria from D. fragilis cultures and for the generation of genome sequence data from D. fragilis. DNA was extracted from human faecal samples and xenic D. fragilis cultures. Extracts were subjected to 16S ribosomal DNA bacterial diversity profiling. Xenic D. fragilis cultures were then subject to antibiotic treatment regimens that systematically removed bacterial species depending on their membrane structure (Gram-positive or Gram-negative) and aerobic requirements. The impact of these treatments on cultures was assessed by 16S amplicon sequencing. Prior to antibiotic treatment, the cultures were dominated by Gram-negative bacteria. Addition of meropenem to cultures eliminated anaerobic Gram-negative bacteria, but it also led to protozoan death after 5 days incubation. The seeding of meropenem resistant Klebsiella pneumoniae strain KPC-2 into cultures before treatment by meropenem prevented death of D. fragilis cells beyond this 5 day period, suggesting that one or more species of Gram-negative bacteria may be an essential nutritional requirement for D. fragilis. Gram-positive cells were completely eliminated using vancomycin without affecting trophozoite growth. Finally, this study shows that genome sequencing of D. fragilis is feasible following bacterial elimination from cultures as the result of the major advances occurring in bioinformatics. We provide evidence on this fact by successfully sequencing the D. fragilis 28S large ribosomal DNA subunit gene using culture-derived DNA.


Assuntos
Dientamoeba/classificação , Dientamoeba/genética , Variação Genética , Genoma de Protozoário/genética , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Fenômenos Fisiológicos Bacterianos , Técnicas de Cultura , Dientamoeba/efeitos dos fármacos , Dientamoeba/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 28S/genética
12.
Sci Rep ; 9(1): 19521, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31863009

RESUMO

Filarial nematode infections cause a substantial global disease burden. Genomic studies of filarial worms can improve our understanding of their biology and epidemiology. However, genomic information from field isolates is limited and available reference genomes are often discontinuous. Single molecule sequencing technologies can reduce the cost of genome sequencing and long reads produced from these devices can improve the contiguity and completeness of genome assemblies. In addition, these new technologies can make generation and analysis of large numbers of field isolates feasible. In this study, we assessed the performance of the Oxford Nanopore Technologies MinION for sequencing and assembling the genome of Brugia malayi, a human parasite widely used in filariasis research. Using data from a single MinION flowcell, a 90.3 Mb nuclear genome was assembled into 202 contigs with an N50 of 2.4 Mb. This assembly covered 96.9% of the well-defined B. malayi reference genome with 99.2% identity. The complete mitochondrial genome was obtained with individual reads and the nearly complete genome of the endosymbiotic bacteria Wolbachia was assembled alongside the nuclear genome. Long-read data from the MinION produced an assembly that approached the quality of a well-established reference genome using comparably fewer resources.


Assuntos
Brugia Malayi/genética , Animais , Feminino , Genoma Mitocondrial/genética , Genoma de Protozoário/genética , Análise de Sequência de DNA
13.
Protist ; 170(6): 125697, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31751779

RESUMO

Desmodesmus costato-granulatus (Skuja) Hegewald 2000 (Sphaeropleales, Chlorophyta) is a small, spineless green alga that is abundant in the freshwater phytoplankton of oligo- to eutrophic waters worldwide. It has a high lipid content and is considered for sustainable production of diverse compounds, including biofuels. Here, we report the draft whole-genome shotgun sequencing of D. costato-granulatus strain SAG 18.81. The final assembly comprises 48,879,637bp with over 4,141 scaffolds. This whole-genome project is publicly available in the CNSA (https://db.cngb.org/cnsa/) of CNGBdb under the accession number CNP0000701.


Assuntos
Clorofíceas/genética , Genoma de Protozoário/genética , Água Doce , Sequenciamento Completo do Genoma
14.
Protist ; 170(6): 125684, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31743821

RESUMO

Hariotina reticulata P. A. Dangeard 1889 (Sphaeropleales, Chlorophyta) is a common member of the summer phytoplankton of meso- to highly eutrophic water bodies with a worldwide distribution. Here, we report the draft whole-genome shotgun sequencing of H. reticulata strain SAG 8.81. The final assembly comprises 107,596,510bp with over 15,219 scaffolds (>100bp). This whole-genome project is publicly available in the CNSA (https://db.cngb.org/cnsa/) of CNGBdb under the accession number CNP0000705.


Assuntos
Evolução Biológica , Clorofíceas/classificação , Clorofíceas/genética , Genoma de Protozoário/genética , Sequenciamento Completo do Genoma
15.
Sci Rep ; 9(1): 17376, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31758058

RESUMO

The genomic sequence of Trypanosoma cruzi, the protozoan causative of Chagas disease was published more than a decade ago. However, due to their complexity, its complete haploid predicted sequence and therefore its genetic repertoire remains unconfirmed. In this work, we have used RNAseq data to improve the previous genome assembly of Sylvio X10 strain and to define the complete transcriptome at trypomastigote stage (mammalian stage). A total of 22,977 transcripts were identified, of which more than half could be considered novel as they did not match previously annotated genes. Moreover, for the first time in T. cruzi, we are providing their relative abundance levels. We have identified that Sylvio X10 trypomastigotes exhibit a predominance of surface protein genes, specifically those encoding trans-sialidase and mucin-like proteins. On the other hand, detailed analysis of the pre-mRNA processing sites revealed some similarities but also some differences in the spliced leader and different polyadenylation addition sites compared to close related kinetoplastid parasites. Our results also confirm that transcription is bidirectional as occur in other kinetoplastids and the proportion of forward-sense and reverse-sense transcripts is almost equivalent, demonstrating that a strand-specificity does not exist.


Assuntos
Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Transcriptoma/fisiologia , Trypanosoma cruzi/genética , Sequência de Bases , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Genoma de Protozoário/genética , Glicoproteínas/genética , Mucinas/genética , Neuraminidase/genética , Poliadenilação/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Análise de Sequência de DNA , Trypanosoma cruzi/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
16.
Parasitol Res ; 118(12): 3195-3204, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31724068

RESUMO

Whole genomic sequencing (WGS) and comparative genomics are increasingly used in the characterization of Cryptosporidium spp. They are facilitated by the establishment of procedures for WGS analysis of clinical specimens without laboratory propagation of pathogens. Results of recent comparative genomics analysis suggest that gene duplication might be associated with broad host ranges of some zoonotic Cryptosporidium species and subtypes, while genetic recombination could be involved in the emergence of virulent subtypes. The availability of WGS data has further facilitated the development of advanced molecular typing tools. The use of these tools together with comparative genomics analyses has begun to improve the investigations of outbreaks in industrialized nations. More WGS data, however, are needed from both industrialized nations and developing countries before we can have in-depth understanding of the population genetics and evolution of Cryptosporidium spp. and genetic determinants of various phenotypic traits in human-pathogenic subtypes.


Assuntos
Criptosporidiose/epidemiologia , Cryptosporidium/genética , Genômica , Animais , Criptosporidiose/parasitologia , Criptosporidiose/transmissão , Cryptosporidium/classificação , Cryptosporidium/patogenicidade , Genoma de Protozoário/genética , Genótipo , Especificidade de Hospedeiro/genética , Humanos , Epidemiologia Molecular , Virulência/genética
17.
Genes (Basel) ; 10(11)2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31752243

RESUMO

In the ciliate Stylonychia, somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for 'RNA-induced DNA replication interference' (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring.


Assuntos
Cilióforos/genética , Regulação da Expressão Gênica no Desenvolvimento , Micronúcleo Germinativo/genética , RNA Mensageiro/biossíntese , RNA Nuclear Pequeno/biossíntese , Replicação do DNA , Genoma de Protozoário/genética , Histonas/genética , Histonas/metabolismo , Micronúcleo Germinativo/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Interferência de RNA , Precursores de RNA/biossíntese , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Telômero/genética , Telômero/metabolismo
18.
BMC Genomics ; 20(1): 744, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619176

RESUMO

BACKGROUND: Clubroot is an important disease of brassica crops world-wide. The causal agent, Plasmodiophora brassicae, has been present in Canada for over a century but was first identified on canola (Brassica napus) in Alberta, Canada in 2003. Genetic resistance to clubroot in an adapted canola cultivar has been available since 2009, but resistance breakdown was detected in 2013 and new pathotypes are increasing rapidly. Information on genetic similarity among pathogen populations across Canada could be useful in estimating the genetic variation in pathogen populations, predicting the effect of subsequent selection pressure on changes in the pathogen population over time, and even in identifying the origin of the initial pathogen introduction to canola in Alberta. RESULTS: The genomic sequences of 43 strains (34 field collections, 9 single-spore isolates) of P. brassicae from Canada, the United States, and China clustered into five clades based on SNP similarity. The strains from Canada separated into four clades, with two containing mostly strains from the Prairies (provinces of Alberta, Saskatchewan, and Manitoba) and two that were mostly from the rest of Canada or the USA. Several strains from China formed a separate clade. More than one pathotype and host were present in all four Canadian clades. The initial pathotypes from canola on the Prairies clustered separately from the pathotypes on canola that could overcome resistance to the initial pathotypes. Similarly, at one site in central Canada where resistance had broken down, about half of the genes differed (based on SNPs) between strains before and after the breakdown. CONCLUSION: Clustering based on genome-wide DNA sequencing demonstrated that the initial pathotypes on canola on the Prairies clustered separately from the new virulent pathotypes on the Prairies. Analysis indicated that these 'new' pathotypes were likely present in the pathogen population at very low frequency, maintained through balancing selection, and increased rapidly in response to selection from repeated exposure to host resistance.


Assuntos
Brassica napus/parasitologia , Genoma de Protozoário/genética , Plasmodioforídeos/genética , Plasmodioforídeos/patogenicidade , Canadá , China , DNA de Protozoário/genética , Resistência à Doença , Variação Genética , Genética Populacional , Filogenia , Doenças das Plantas/parasitologia , Plasmodioforídeos/classificação , Seleção Genética , Análise de Sequência de DNA , Estados Unidos
19.
Mem Inst Oswaldo Cruz ; 114: e190147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553371

RESUMO

BACKGROUND: Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES: Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS: In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION: This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.


Assuntos
Calpaína/genética , Genoma de Protozoário/genética , Leishmania braziliensis/química , Macrófagos Peritoneais/metabolismo , Animais , Western Blotting , Calpaína/efeitos dos fármacos , Calpaína/metabolismo , Calpaína/ultraestrutura , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica , Imuno-Histoquímica , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Virulência
20.
Mol Plant Microbe Interact ; 32(12): 1559-1563, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31479390

RESUMO

Resolving complex plant pathogen genomes is important for identifying the genomic shifts associated with rapid adaptation to selective agents such as hosts and fungicides, yet assembling these genomes remains challenging and expensive. Phytophthora capsici is an important, globally distributed plant pathogen that exhibits widespread fungicide resistance and a broad host range. As with other pathogenic oomycetes, P. capsici has a complex life history and a complex genome. Here, we leverage Oxford Nanopore Technologies and existing short-read resources to rapidly generate a low-cost, improved assembly. We generated 10 Gbp from a single MinION flow cell resulting in >1.25 million reads with an N50 of 13 kb. The resulting assembly is 95.2 Mbp in 424 scaffolds with an N50 length of 313 kb. This assembly is approximately 30 Mbp bigger than the current reference genome of 64 Mbp. We confirmed this larger genome size using flow cytometry, with an estimated size of 110 Mbp. BUSCO analysis identified 97.4% complete orthologs (19.2% duplicated). Evolutionary analysis supports a recent whole-genome duplication in this group. Our work provides a blueprint for rapidly integrating benchtop long-read sequencing with existing short-read data, to dramatically improve assembly quality and integrity of complex genomes and offer novel insights into pathogen genome function and evolution.


Assuntos
Genoma de Protozoário , Phytophthora , Análise de Sequência de DNA , Tamanho do Genoma , Genoma de Protozoário/genética , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Phytophthora/genética
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