Major changes in chromosomal somy, gene expression and gene dosage driven by SbIII in Leishmania braziliensis and Leishmania panamensis

Abstract

Leishmania braziliensis and Leishmania panamensis are two species clinically and epidemiologically important, among others because of their relative resistance to first-line drugs (antimonials). The precise mechanism underlying the ability of these species to survive antimony treatment remains unknown. Therefore, elucidating the pathways mediating drug resistance is essential. We herein experimentally selected resistance to trivalent antimony (Sb^III) in the reference strains of L. braziliensis (MHOM/BR75/M2904) and L. panamensis (MHOM/COL/81L13) and compared whole genome and transcriptome alterations in the culture promastigote stage. The results allowed us to identify differences in somy, copy number variations in some genes related to antimony resistance and large-scale copy number variations (deletions and duplications) in chromosomes with no somy changes. We found mainly in L. braziliensis, a direct relation between the chromosomal/local copy number variation and the gene expression. We identified differentially expressed genes in the resistant lines that are involved in antimony resistance, virulence, and vital biological processes in parasites. The results of this study may be useful for characterizing the genetic mechanisms of these Leishmania species under antimonial pressure, and for clarifying why the parasites are resistant to first-line drug treatments.

Figure 1

Dynamics of ploidy in L. braziliensis and L. panamensis. Comparisons of the chromosomal copy number between lines sensitive and resistant to Sb^III in L. braziliensis (left) and L. panamensis (right). The grey points indicate the chromosomes that underwent a change in somy.

Figure 2

Intrachromosomal duplication in Sb^III-resistant L. panamensis mutants. Raw read depth for chromosome 27 of the Sb^III-resistant and -sensitive lines. The black and gray lines show the raw depth in Lp_SSG_S and Lp_SSG_R lines, respectively. The figure below is a representation of genes located in this region.

Figure 3

Gene copy number variation between sensitive and resistant lines of L. braziliensis and L. panamensis. Selection of genes with different copy numbers in the SSG_S and SSG_R lines (difference >1.0) in (A) L. braziliensis and (B) L. panamensis. The genes in L. panamensis were listed along with their corresponding L. braziliensis ortholog genes.

Figure 4

Single nucleotide polymorphism (SNPs) in the chromosome 32 of L. braziliensis sensitive and resistant to Sb^III. The figure shows the SNPs (DNA/RNA) found in the chromosome 32, comparing the resistant and sensitive lines to Sb^III. The figure below is a representation of the gene transcription in chr 32.

Figure 5

Relationship between chromosomal copy number variations and gene expression levels in the SSG_S and SSG_R lines of L. braziliensis and L. panamensis. The heatmaps show median normalized read depths of 35 chromosomes (y axis) found in L. braziiensis (Left) and L. panamensis (right) sensitive and resistant to Sb^III (x axis) The color key indicates the somy value (S), which ranged from 1 to 5 as follows: monosomy, S < 1.5; disomy, 1.5 ≤ S < 2.5; trisomy, 2.5 ≤ S < 3.5; tetrasomy, 3.5 ≤ S < 4.5; and pentasomy, 4.5 ≤ S < 5. A black triangle in an upper right corner indicates a significant change in S value. R (replicates).

Figure 6

Transcriptional profile of the SSG_R and SSG_S lines in L. braziliensis and L. panamensis. (A) Venn diagram showing transcripts down- and up-regulated more than 2-fold in the resistant lines regarding the expression levels found in the parental sensitive line. The number of transcripts having significantly altered levels (up and down regulated) and the number of transcripts with DE when both species were compared (middle of each diagram) (B,C) Differentially expressed transcripts between the experimental conditions. B (L. braziliensis). C (L. panamensis). The graphic in the middle represents the MA plot constructed based on the DESeq2 results. Red points indicate significance at a 10% adjusted p-value, up-regulation and down-regulation respectively. Grey triangles indicate transcripts that showed no change. The lower figures present the results of the Gene Ontology enrichment analyses for biological process of up (left) and down (right) regulated transcripts. The transcripts included had an expression value higher than 2-fold change in both comparisons. The number of transcripts with a GO term is indicated in the corresponding pie slice.