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

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

Conservation of large syntenic groups between the G strain and clone D11.

<p>Selected markers belonging to <i>in silico</i> chromosomes TcChr37 (<b>Panel A</b>) and TcChr39 (<b>Panel B</b>) previously defined in clone CL Brener were mapped on chromosomal bands of the G strain and clone D11 separated by PFGE. The diagrammatic representation above each panel indicates the position of the markers on the <i>in silico</i> chromosome. Markers from TcChr37 are THTc, TEUF0001, TEUF0180 and delta-6. Markers from TcChr39 are XM_811753, H49, JL8 and ankyrin. 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

Identification of possible chromosomal rearrangements in clone D11.

<p>Mapping of markers belonging to <i>in silico</i> chromosomes TcChr7 (<b>Panel A</b>) and TcChr22 (<b>Panel B</b>). Identification of chromosomal rearrangements involving one band in the G strain and two bands in clone D11 (<b>Panel A</b>) or vice versa (<b>Panel B</b>). The positions of the markers used as radiolabeled probes are indicated in the diagrammatic representation of the <i>in silico</i> chromosomes. Markers from TcChr7 are XM_799116, XM_803657, TryP and NLI. Markers from TcChr22 are XM_801648, XM_801647, Bpp-1 and XM_801649. 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

Telomere length polymorphism of the G strain and clone D11.

<p><b>Panel A</b>) Southern-blot hybridization of restriction fragments generated by <i>Hae</i>III and <i>Msp</i>I probed with the telomeric repeat (TTAGGG). <i>Hae</i>III-digested phage lambda DNA (used as a molecular weight marker) is shown on the left. <b>Panel B</b>) Analysis of the subtelomeric length of the G strain and clone D11 chromosomes was performed by Southern-blot hybridization of <i>Sfi</i>I restriction fragments with the telomeric repeat. The size of the larger subtelomeric fragment of clone D11 is shown on the left.</p

Possible mechanisms of genetic recombination that could give rise to chromosomal polymorphism in <i>T.</i><i>cruzi</i>.

<p><b>Panel A</b>) Translocation mechanism: a DNA fragment (210 kb) from a heterologous chromosome (red) is translocated to another chromosome (blue) by homologous recombination, generating “homologous” chromosomes of different sizes. <b>Panel B</b>) Fusion and breakage mechanism: two homologous chromosomes of different sizes are fused, forming a dicentric chromosome which is then broken, generating two chromosomes of similar sizes but with different gene content.</p

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

Karyotype polymorphism between the G strain and clone D11.

<p><b>Panel A)</b> Chromosomal bands were separated by Pulsed-Field Gel Electrophoresis (PFGE) and stained with SYBR Green I. The bands from the G strain were numbered using Arabic numerals (1–19) as in a previous study (Souza et al., 2011) while capital letters (A – U) were used for clone D11, starting from the smallest band. <b>Panel B)</b> Diagrammatic representation of the molecular karyotypes of the G strain and clone D11. The rectangles represent a unique distinguishable band visualized after SYBR Green I staining. The thickness of the rectangles represents the proportional staining of each chromosomal band. The number and letter of chromosomal bands as well as their molecular weight are indicated to the left and right of each strip, respectively.</p