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. 2018 Nov 6;9(6):e01399-18.
doi: 10.1128/mBio.01399-18.

Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification

Affiliations

Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification

Giovanni Bussotti et al. mBio. .

Abstract

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.

Keywords: Leishmania; aneuploidy; evolution; gene copy number variation; genomic adaptation; telomeric amplification.

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Figures

Fig. 1
Fig. 1
SNVs and translocations with respect to the reference genomes. Venn diagrams show the number of unique and shared SNVs among three L. infantum strains (A), three L. donovani strains (B), and two L. major strains together with an L. tropica strain (C). (D) Circos representation of genomic translocations in samples Ldo_CH33 and Ldo_LTB compared to the corresponding L. donovani reference genome. Connecting lines represent translocations events. Black and red lines demonstrate, respectively, Ldo_CH33 and Ldo_LTB specific translocations. Blue lines show translocations common in both strains. No inversions were detected using the filtering settings indicated in the Materials and Methods section. Black, chromosomes; red, genes mapping on the positive strand; green, genes mapping on the negative-strand.
Fig. 2
Fig. 2
Interstrain gene CNV. (A to C) Ternary plots showing for each gene the relative abundance in the three considered strains (left panels). The axes report the fraction of the normalized gene coverage in the three strains, with each given point adding up to 100. Black dots represent unique genes, whereas red dots indicate genes representing gene families. Comparisons of three L. infantum strains (A), three L. donovani strains (B), and two L. major strains together with an L. tropica strain (C) are shown. The right panels show examples of detected gene copy number variations (CNVs). From top to the bottom, the tracks represent the sequencing depth measured in the three strains, the gene annotations, and the predicted repetitive elements. Coverage tracks were produced with bamCoverage from the deepTools suite (48) (version 2.4.2), ignoring duplicated reads. Normalization of reads per kilobase per million (RPKM) was applied to render the coverage comparable across samples.
Fig. 3
Fig. 3
Chromosome ploidy analysis. Box plots represent the normalized sequencing coverage distributions for each chromosome for the strains indicated. The lower and upper edges of the box show, respectively, the lower quartile (i.e., 25% of nucleotides with normalized coverage below that value) and upper quartile (i.e., 25% of nucleotides with normalized coverage above that value). The whiskers show maximum and minimum coverage values, excluding outliers. Outliers are not shown to ease plot readability. Box sizes reflect coverage dispersion that can be affected by sample sequencing depth, chromosomal ploidy, intrachromosomal copy number alterations, assembly gaps, or repetitive regions. The increased box size visible in chromosome 27 of sample Ldo_LTB is caused by a large subchromosomal amplification (Fig. S3). In L. donovani, L. major, or L. tropica samples, the presence of large gaps or repetitive regions inflates the box size for chromosomes 2, 8, and 12. Green, early passage (EP); orange, EP + 3.1 replicate; purple, EP + 3.2 replicate.
Fig. 4
Fig. 4
Gene copy number variation (CNV) during culture adaptation. (A) Genome-wide scatter plot showing log10 gene coverage of EP and EP + 3 samples. Dots represent all genes annotated in the respective reference assemblies. (B) Chromosome-specific scatter plots of gene CNVs between EP + 3 versus EP. Only selected chromosomes are shown, and the full panel is available in Fig. S4. The red diagonal lines indicate the bisectors. The gray dashed horizontal lines mark a coverage value of 1. The axes’ maximum and minimum values were adjusted to the most extreme values for each plot to avoid logarithmic compression. For both panels A and B, the EP + 3.1 replicate was used, except for Lmj_A445, for which the EP + 3.2 replicate was utilized.
Fig. 5
Fig. 5
Subtelomeric amplification. (A) Genome-wide coverage ratios (y axes) between EP and EP + 3 of the indicated samples and their respective reference genomes (left and middle panels) or between EP + 3/EP (right panels) are shown. The EP + 3 coverage refers to the EP + 3.1 replicate, except for Lmj_A445, for which EP + 3.2 replicate coverage was used. The x axis reports the position of the genomic windows along the chromosomes. Dots represent genomic windows of 300 bases. In each panel, the 36 Leishmania chromosomes are shown in sequential order. To ease the visualization, all scores of >3 were assigned to a value of 3. (B) The EP + 3/EP coverage ratio for chromosomes 3, 7, and 13 of sample Linf_02A (top panel) and the Integrative Genomics Viewer (IGV) snapshots of the respective chromosome extremities (bottom panel) are shown. The lower tracks (in order of appearance from the top) correspond to sequencing coverage in EP, sequencing coverage in EP + 3, repeat elements, or predicted low-complexity region predictions and L. infantum gene annotations. The sequencing coverage tracks range from 0 to ×ばつ. For chromosomes 7 and 13, the bottom panels highlight in orange the misassembled regions. (C) SyntView snapshot of chromosomes 7 and 13. From top to bottom, the tracks show the orthologous genes in L. infantum JPCM5, L. donovani BPK282A1, L. donovani PBQ71C8, and L. major Friedlin. Straight lines connect the orthologous genes in different genomes. The diagonal lines are indicative of misassembled genomic regions.

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