Subtelomeric I-Scel-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi

Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a I cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-Scel meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Centroccidental Lisandro Alvarado, Lab Genet Mol Dr Yunis Turbay, Ciencias Salud, Barquisimeto, VenezuelaNIAID, Lab Malaria & Vector Res, NIH, Rockville, MD USAUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilConsejo Nacl Invest Cient & Tecn, Inst Invest Ingn Genet & Biol Mol, Lab Biol Mol Enfermedad Chagas, Buenos Aires, DF, ArgentinaJ Craig Venter Inst, Dept Infect Dis, Rockville, MD USAFdn Inst Estudios Avanzados, Ctr Biotecnol, Caracas, VenezuelaUniv Estadual Campinas, Fac Ciencias Med, Dept Patol Clin, Campinas, SP, BrazilDepartamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, BrazilFAPESP: 11/51693-0FAPESP: 11/51475-3CNPq: 306591/2015-4Web of Scienc

Anatomy and evolution of telomeric and subtelomeric regions in the human protozoan parasite Trypanosoma cruzi

Background: the subtelomeres of many protozoa are highly enriched in genes with roles in niche adaptation. T. cruzi trypomastigotes express surface proteins from Trans-Sialidase (TS) and Dispersed Gene Family-1 (DGF-1) superfamilies which are implicated in host cell invasion. Single populations of T. cruzi may express different antigenic forms of TSs. Analysis of TS genes located at the telomeres suggests that chromosome ends could have been the sites where new TS variants were generated. the aim of this study is to characterize telomeric and subtelomeric regions of T. cruzi available in TriTrypDB and connect the sequences of telomeres to T. cruzi working draft sequence.Results: We first identified contigs carrying the telomeric repeat (TTAGGG). of 49 contigs identified, 45 have telomeric repeats at one end, whereas in four contigs the repeats are located internally. All contigs display a conserved telomeric junction sequence adjacent to the hexamer repeats which represents a signature of T. cruzi chromosome ends. We found that 40 telomeric contigs are located on T. cruzi chromosome-sized scaffolds. in addition, we were able to map several telomeric ends to the chromosomal bands separated by pulsed-field gel electrophoresis. the subtelomeric sequence structure varies widely, mainly as a result of large differences in the relative abundance and organization of genes encoding surface proteins (TS and DGF-1), retrotransposon hot spot genes (RHS), retrotransposon elements, RNA-helicase and N-acetyltransferase genes. While the subtelomeric regions are enriched in pseudogenes, they also contain complete gene sequences matching both known and unknown expressed genes, indicating that these regions do not consist of nonfunctional DNA but are instead functional parts of the expressed genome. the size of the subtelomeric regions varies from 5 to 182 kb; the smaller of these regions could have been generated by a recent chromosome breakage and telomere healing event.Conclusions: the lack of synteny in the subtelomeric regions suggests that genes located in these regions are subject to recombination, which increases their variability, even among homologous chromosomes. the presence of typical subtelomeric genes can increase the chance of homologous recombination mechanisms or microhomology-mediated end joining, which may use these regions for the pairing and recombination of free ends.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFIOCRUZ MG, Ctr Pesquisas Rene Rachou, Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, ICB, Dept Parasitol, Belo Horizonte, MG, BrazilUCLA, Barquisimeto, VenezuelaFdn Inst Estudios Avanzados IDEA, Caracas, VenezuelaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Análise genômica comparativa de um clone de trypanosoma cruzi e a cepa parental

The taxon Trypanosoma cruzi consists of genetically heterogeneous populations that differ both in genotypic aspects and phenotypic adaptations and virulence towards the mammalian host. The genetic variability is also reflected in the genome organization. Iinterand intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cellderived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. In a previous work, we have demonstrated the existence of phenotypic, genotypic and karyotypic differences between clone D11 and the parental G strain. Here we performed intraspecific comparative genomic hybridizations to identify chromosome regions harboring copy number variants in the clone D11 and G strain. The most commonly observed aberrations in the clone D11 were deletions and duplications of small chromosomal segments (<50 kb) which could be mediated by homologous recombination. Most of variants was related to loss of DNA and the unequal distribution of genes between the regions of loss or gain of DNA suggest that multigene families can be involved in recombination events between G strain and clone D11. The karyotypes of clone D11 and G strain differ in both the number and size of the chromosome bands. The chromosomal rearrangements detected by hybridization of chromoblots with chromosome-specific markers were confirmed by aCGH. Based on the aCGH data we suggest mechanisms of recombination to explain the chromosomal rearrangements. Genomic changes detected by aCGH suggest the presence of a dynamic genome that respond to environmental stress by varying the number of gene copies and its distribution in the chromosome. These changes could be present in the original population, a multiclonal population, or have been induced by stress in cloning in a monoclonal population. Our data support the hypothesis of a multiclonal population in T. cruzi. However, current evidence suggests the predominance of segmental aneuploidy in T. cruzi, i.e. involving parts of the chromosome, while in Leishmania events involving the entire chromosome (chromosome aneuploidy) are the most common.O táxon Trypanosoma cruzi consiste em populações geneticamente heterogêneas que diferem tanto em aspectos genotípicos, adaptações fenotípicas e virulência em relação ao hospedeiro mamífero. A variabilidade genética também reflete na organização do genoma. Variações cariotípicas inter- e intra-cepa tem sido relatadas, sugerindo que rearranjos cromossômicos ocorreram durante a evolução deste parasita. O clone D11 é um clone derivado de uma única célula da cepa G de T. cruzi, selecionado pelo método de diluição mínima e por infecção de células Vero com tripomastigotas metacíclicos. Em um trabalho anterior, nós demonstramos a existência de diferenças fenotípicas, genotípicas e cariotípicas entre clone D11 e a cepa parental G. Nós aplicamos a hibridização genômica comparativa intraespecífica para identificar regiões cromossômicas com variações do número de cópias genicas entre o clone D11 e a cepa G. As alterações mais comumente observadas no clone D11 foram deleções e duplicações de pequenos segmentos cromossômicos (<50 kb) que poderiam ser mediadas por recombinação homóloga. A maioria das alterações foi relacionada com a perda de DNA e a distribuição desigual de genes entre as regiões de perda ou ganho de DNA sugerem que as famílias multigênicas podem estar envolvidas em eventos de recombinação entre a cepa G e o clone D11. Os cariótipos do clone D11 e da cepa G diferem em número e tamanho das bandas cromossômicas. Os rearranjos cromossômicos no clone D11 detectados pela hibridização de ?chromoblots? com marcadores cromossomo-específicos foram confirmados aCGH. Baseando-se nos dados de aCGH nós propomos mecanismos de recombinação para explicar os rearranjos cromossômicos. As alterações genômicas detectadas por aCGH sugerem a presença de um genoma dinâmico que responde à pressão ambiental variando o número de cópias genicas e sua distribuição no cromossomo. Estas alterações poderiam estar presentes em na população original, ou seja, uma população multiclonal, ou foram induzidas pelo estresse causado na clonagem em uma população monoclonal. Nossos dados apoiam a hipótese de uma população multiclonal em T. cruzi. Porém, as evidências atuais sugerem predominância de aneuploidia segmentada em T. cruzi, isto é, envolvendo partes do cromossomo, enquanto em Leishmania eventos envolvendo todo o cromossomo (aneuploidia cromossômica) são os mais comuns.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Interclonal Variations in the Molecular Karyotype of <i>Trypanosoma cruzi</i>: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

<div><p><i>Trypanosoma cruzi</i> comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the <i>T. cruzi</i> G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for <i>Leishmania infantum,</i> have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the <i>T. cruzi</i> arsenal for responding to environmental pressure.</p></div

Location of molecular markers on G strain and clone D11 chromosomal bands.

#<p>Chromosomal bands separated by PFGE and stained with ethidium bromide.</p

Identification of a rearrangement involving a large fragment containing the α- tubulin gene in clone D11.

<p><b>Panel A</b>) Mapping of the α-tubulin gene on chromosomal bands of the G strain and clone D11 showing a translocation event involving large chromosomes. β-tubulin, hypothetical protein XM_804243 and endomembrane protein (XM_ 812238) were also mapped and showed the same hybridization profile. The positions of markers used as probes are indicated in the diagrammatic representation of in silico chromosomes TcChr14. <b>Panel B</b>) Restriction fragment analysis of α-tubulin gene loci was carried out by digesting genomic DNA with <i>Pst</i>I (P) or double-digesting it with <i>Bgl</i>II and <i>Pst</i>I (B/P). Phage lambda DNA digested with <i>Hae</i>III, used as a molecular weight marker, is shown on the left. <b>Panel C</b>) Restriction analysis of whole chromosomes in agarose blocks was performed using the rare-cutting enzymes <i>Pac</i>I and <i>Sfi</i>I. The molecular weights of fragments recognized by the probe are shown on the left.</p

Identification of homologous chromosomal bands of similar molecular sizes in the G strain and clone D11.

<p>Hybridization profile of specific chromosomal markers hybridized to one or more bands of similar molecular size in both isolates after chromosome separation by PFGE and Southern-blot hybridization. The markers used are TEUF0099, rDNA18S, TEUF0242 and ADC. Gene identification and GenBank accession number of each marker are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063738#pone-0063738-t001" target="_blank">Table 1</a>.</p

Allele sizes (bp) for each microsatellite locus amplified for the G strain and clone D11.

a<p>Microsatellite loci with the same alleles in the G strain and D11 clone.</p>b<p>Microsatellite loci with a common allele in the G strain and D11 clone.</p>c<p>Microsatellite loci with different alleles in the G strain and D11 clone.</p