Gene Expression in Leishmania Is Regulated Predominantly by Gene Dosage

Abstract

Leishmania tropica, a unicellular eukaryotic parasite present in North and East Africa, the Middle East, and the Indian subcontinent, has been linked to large outbreaks of cutaneous leishmaniasis in displaced populations in Iraq, Jordan, and Syria. Here, we report the genome sequence of this pathogen and 7,863 identified protein-coding genes, and we show that the majority of clinical isolates possess high levels of allelic diversity, genetic admixture, heterozygosity, and extensive aneuploidy. By utilizing paired genome-wide high-throughput DNA sequencing (DNA-seq) with RNA-seq, we found that gene dosage, at the level of individual genes or chromosomal "somy" (a general term covering disomy, trisomy, tetrasomy, etc.), accounted for greater than 85% of total gene expression variation in genes with a 2-fold or greater change in expression. High gene copy number variation (CNV) among membrane-bound transporters, a class of proteins previously implicated in drug resistance, was found for the most highly differentially expressed genes. Our results suggest that gene dosage is an adaptive trait that confers phenotypic plasticity among natural Leishmania populations by rapid down- or upregulation of transporter proteins to limit the effects of environmental stresses, such as drug selection.IMPORTANCELeishmania is a genus of unicellular eukaryotic parasites that is responsible for a spectrum of human diseases that range from cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) to life-threatening visceral leishmaniasis (VL). Developmental and strain-specific gene expression is largely thought to be due to mRNA message stability or posttranscriptional regulatory networks for this species, whose genome is organized into polycistronic gene clusters in the absence of promoter-mediated regulation of transcription initiation of nuclear genes. Genetic hybridization has been demonstrated to yield dramatic structural genomic variation, but whether such changes in gene dosage impact gene expression has not been formally investigated. Here we show that the predominant mechanism determining transcript abundance differences (>85%) in Leishmania tropica is that of gene dosage at the level of individual genes or chromosomal somy.

Keywords: CNV; Leishmania; RNA-seq; gene dosage; gene expression.

FIG 1

Heterozygosity and homozygosity among a total of 268,518 polymorphic sites identified by analyzing the genomes of 14 isolates of L. tropica. (A) Heterozygosity and homozygosity among the total 268,518 polymorphic positions in 14 isolates of L. tropica. The F statistic (inbreeding coefficient) and the number of heterozygous (He) and homozygous (Ho) positions were calculated using VCFtools. The isolates were sorted into low, intermediate, and high homozygosity groups, depending on the Ho. (B) LROH in 14 clinical isolates of L. tropica. The RCircos plot shows LROH for each of the 36 chromosomes, with homozygosity in blue and heterozygosity in gold. Note that no two isolates had the same exact pattern of LROH, despite many similarities among individual geographic groups (several isolates from Israel and Jordan were mostly homozygous, whereas the majority of the other isolates were largely heterozygous). The isolates represented in each panel are ordered in concentric rings, according to the F value (inbreeding coefficient) from panel A and numbered from 1 to 14, respectively: 1, K112_1 (India); 2, Rupert, Afghanistan; 3, KK27 (Afghanistan); 4, Azad, Afghanistan; 5, Kubba (Syria); 6, Ackerman (Israel); 7, Boone, Saudi Arabia; 8, Melloy (Saudi Arabia); 9, K26_1 (India); 10, MN-11 (Jordan); 11, L747 (Israel); 12, MA-37 (Jordan); 13, L810 (Israel); 14, E50 (Israel).

FIG 2

Heterozygous allele frequencies and read depths for selected chromosomes of two L. tropica isolates, K26 (India) and Ackerman (Israel). (A) K26 (chromosomes 23, 24, and 28); (B) Ackerman (chromosomes 9, 11, and 24). Variants called homozygous differences from the L590 reference scored either 1 or 0 and are depicted in the 0.1 or 0.9 y axis range. Variants called heterozygous show allele frequencies around 0.5 in the hybrid line for all disomic chromosomes. Variants called heterozygous on trisomic chromosomes show allele frequencies around 0.33 and 0.67 in the hybrid line. Allele frequency and the total number of biallelic SNPs are shown in the upper panel, and read depth across the chromosome is shown in the lower panel for each chromosome. Median read depth, marked by a red line, was approximately 50 reads for disomic chromosomes.

FIG 3

Genome-wide F_ST estimates and localized phylogenetic analysis indicated extensive genetic hybridization among isolates of L. tropica. (A) Pairwise F_ST estimates indicate localized high intrapopulation genetic diversity within the population of L. tropica clinical isolates that were predominantly homozygous (referred to as Pop1 in the main text). F_ST estimates were obtained with VCFtools, comparing isolates from Jordan (MA-37 and MN-11) to isolates from Israel (E50, LRC-L810, and LRC-L747). Regions surrounding peaks in F_ST values are highlighted in the plot as follows: a, LmjF.08 381000 to 439000; b, LmjF.08, 439000 to 550000; c, LmjF.35 1168000 to 1396000; d, LmjF.35 1396000 to 1624000; e, LmjF.35 1624000 to 1852000. These regions were chosen to generate phylogenetic trees to determine the allelic diversity present and its inheritance patterns across the homozygous strains. The y axis shows the pairwise F_ST values, whereas the x axis indicates the positions of the 36 chromosomes. Each dot represents the F_ST value in a 1-kb window. Separate phylogenetic trees using the SNP information from regions a to e reflect a swapping of ancestral haploblocks among the homozygous populations of L. tropica. Neighbor-joining phylogenetic trees were constructed using MEGA with 50% majority rules. (B) F_ST plot showing the genome-wide pairwise F_ST values identified when the homozygous population (n = 5; E50, LRC-L810, MA-37, LRC-L747, and MN-11) was compared against all other isolates that were heterozygous (n = 9; K112_1, Rupert, KK27, Azad, Kubba, Ackerman, K26_1, Boone, and Melloy). x and y axes are labeled as described above, and each dot represents the F_ST value in a 1-kb window. As expected, the F_ST values are higher for comparison of these two very divergent populations. (C) F_ST plot showing the genome-wide pairwise F_ST values identified within the heterozygous population comparing those that were heterozygous throughout (n = 5; K112_1, Rupert, KK27, Azad, and Kubba) with those that were heterozygous but possessed LROH (n = 4; Ackerman, K26_1, Boone, and Melloy). x and y axes are labeled as described above, and each dot represents the F_ST value in a 1-kb window. High localized F_ST values again suggest extensive haploblock swapping, as in panel A.

FIG 4

Extensive aneuploidy in 14 geographically diverse isolates of L. tropica. Estimated somy at each of the 36 chromosomes in the L. tropica genome, with increasing somy indicated from yellow (somy = 2), to orange (somy = 3), to red (somy = 4).

FIG 5

Isolate L810 (Israel) showed an aberrant expression signature compared to all other isolates. (A) PCA plot of expression data, showing the first two principal components on the x and y axes for all strains except Boone (one of the three replicates showed some characteristics of an outlier; see the main text). Each set of triplicates is represented in a different color, depending on the isolate of origin. Note the close clustering of each set of triplicates, indicating accurate biological replication. The replicates for isolate L810 clustered closely together, but quite distantly from the rest of the isolates. (B) Heat map of Euclidean distances between variance-stabilized expression values for each pair of samples, with larger Euclidean distances represented by darker shades of blue. Expression signatures within each set of triplicates were very similar to each other (represented in light colors along the diagonal axis). Isolate L810 showed an aberrant expression signature, with higher distance values in all pairwise comparisons (average distance values were closer to 50, instead of 33, as with the rest of the isolates).

FIG 6

The pairwise comparison between K26 (India) and Ackerman (Israel) detected gene dosage effects due to copy number variation and somy differences. (A) Somy. The top graph represents the log fold changes in expression at genes along the chromosome, whereas the bottom graph represents the log scale ratios between the read depths in K26 (upper quadrant) and Ackerman (lower quadrant). The x axis represents base pair positions along the chromosome in both graphs. (B) Gene dosage effects due to copy number variation on the transcription of genes. Three large CNVs spanning 10 or more DE genes on chromosome 23, 24, and 27 are shown (CNVR159-178 on chromosome 27, CNVR114 on chromosome 24, and CNVR240 on chromosome 23) (Table S5). A smaller CNV on chromosome 24, upstream of the larger CNV, and one in the middle of chromosome 20 are also shown. Gene dosage effects in the two CNVs on chromosome 24 appear to behave in a dose-dependent fashion, with a 2-fold increase in copy number (consistent with a homozygous duplication) leading to a 2-fold increase in expression and a 1-fold increase in copy number (consistent with a heterozygous duplication), leading to a 1-fold increase in expression. The CNV on chromosome 20 does not appear to impact gene expression.

FIG 7

Global changes in transcription highlight significant gene dosage effects, with differential expression of membrane-bound transporter genes in a pairwise comparison between K26 (India) and Ackerman (Israel). (A) RCircos plot illustrating gene dosage effects on transcription genome-wide due to aneuploidy in a pairwise comparison between K26 and Ackerman. These isolates were selected due to the large number of chromosomes that differed for somy between each other. Tracks 1 and 3 represent somy for K26 and Ackerman, respectively, while the results shown in track 2 were obtained using edgeR to graphically represent differentially expressed genes, with upregulated genes in red (log fold change > 0) and downregulated genes in blue (log fold change < 0). Log fold changes in expression mirror differences in somy, indicating substantial gene dosage effects in L. tropica. (B) Heat map of the 30 most significantly differentially expressed genes, represented with their log fold changes in expression. A darker shade of blue indicates higher expression. Note the large cluster of DE genes on chromosome 24 that were upregulated in isolates K26 and MN11. This cluster is part of the CNV in isolate K26 (shown in Fig. 6; CNVR114). Other notable genes include LmjF.35.5150 and LmJF.10.0385, which appear to be amplified and deleted, respectively, in K26. These genes code for a biopterin transporter and a folate transporter that are known to act in concert in folate metabolism. A gene encoding a hypothetical MFS general transporter (LmjF.11.0680) was also upregulated in isolates Melloy and Ackerman.