Genomic and Transcriptomic Analysis for Identification of Genes and Interlinked Pathways Mediating Artemisinin Resistance in Leishmania donovani

Abstract

Current therapy for visceral leishmaniasis (VL), compromised by drug resistance, toxicity, and high cost, demands for more effective, safer, and low-cost drugs. Artemisinin has been found to be an effectual drug alternative in experimental models of leishmaniasis. Comparative genome and transcriptome analysis of in vitro-adapted artesunate-resistant (K133AS-R) and -sensitive wild-type (K133WT) Leishmania donovani parasites was carried out using next-generation sequencing and single-color DNA microarray technology, respectively, to identify genes and interlinked pathways contributing to drug resistance. Whole-genome sequence analysis of K133WT vs. K133AS-R parasites revealed substantial variation among the two and identified 240 single nucleotide polymorphisms (SNPs), 237 insertion deletions (InDels), 616 copy number variations (CNVs) (377 deletions and 239 duplications), and trisomy of chromosome 12 in K133AS-R parasites. Transcriptome analysis revealed differential expression of 208 genes (fold change ≥ 2) in K133AS-R parasites. Functional categorization and analysis of modulated genes of interlinked pathways pointed out plausible adaptations in K133AS-R parasites, such as (i) a dependency on lipid and amino acid metabolism for generating energy, (ii) reduced DNA and protein synthesis leading to parasites in the quiescence state, and (iii) active drug efflux. The upregulated expression of cathepsin-L like protease, amastin-like surface protein, and amino acid transporter and downregulated expression of the gene encoding ABCG2, pteridine receptor, adenylatecyclase-type receptor, phosphoaceylglucosamine mutase, and certain hypothetical proteins are concordant with genomic alterations suggesting their potential role in drug resistance. The study provided an understanding of the molecular basis linked to artemisinin resistance in Leishmania parasites, which may be advantageous for safeguarding this drug for future use.

Keywords: Leishmania donovani; artemisinin drug resistance; transcriptome; whole-genome sequencing (WGS).

Figure 1

Comparative Analysis of Single Nucleotide Polymorphisms (SNPs) present in K133AS-R with K133WT. (A) Venn diagram showing the unique genes present in K133WT and K133AS-R. (B) Comparative SNP density analysis of K133WT vs. K133AS-R (C) Pie chart showing the percentage of different gene variants present in K133WT and K133AS-R.

Figure 1

Comparative Analysis of Single Nucleotide Polymorphisms (SNPs) present in K133AS-R with K133WT. (A) Venn diagram showing the unique genes present in K133WT and K133AS-R. (B) Comparative SNP density analysis of K133WT vs. K133AS-R (C) Pie chart showing the percentage of different gene variants present in K133WT and K133AS-R.

Figure 2

Analysis of CNV diversity in artesunate-resistant L.donovani (K133AS-R). (A) Size distribution of CNVs detected in the K133AS-R genome (B) Comparative CNV analysis of K133WT vs. K133AS-R.

Figure 3

Chromosomy estimation in L. donovani parental line (K133WT) and artemisinin-resistant lines K133AS-R. The solid line represents median coverage and it was assigned a value of 2, considering that diploid is the principal ploidy state in Leishmania. The dotted line represents the calculated values for other somies (blue- monosomy; between two dotted red-trisomy).

Figure 4

Characterization of K133WT and K133AS-R unigenes based on an NCBI non redundant (Nr) protein database search. (A) Species distribution of the top Blast hits for the K133WT assembled unigenes and (B) Species distribution of the top Blast hits for the K133AS-R assembled unigenes with a cutoff E-value of 10⁻⁰⁵. Gene Ontology (GO) annotation for all the assembled unigenes in K133WT (C) and K133AS-R (D) GO-terms were assigned to functionally annotate the genes based on BLAST search results using the Blast2GO program (Biobam BioInformatics, Valencia, Spain). The results were classified based in three functional categories, Green bar represents biological function (BF); Blue: molecular function (MF); and Yellow: cellular component (CC).

Figure 4

Characterization of K133WT and K133AS-R unigenes based on an NCBI non redundant (Nr) protein database search. (A) Species distribution of the top Blast hits for the K133WT assembled unigenes and (B) Species distribution of the top Blast hits for the K133AS-R assembled unigenes with a cutoff E-value of 10⁻⁰⁵. Gene Ontology (GO) annotation for all the assembled unigenes in K133WT (C) and K133AS-R (D) GO-terms were assigned to functionally annotate the genes based on BLAST search results using the Blast2GO program (Biobam BioInformatics, Valencia, Spain). The results were classified based in three functional categories, Green bar represents biological function (BF); Blue: molecular function (MF); and Yellow: cellular component (CC).

Figure 5

Comparative transcriptome profiling of K133WT and K133AS-R isolate. (A) Comparative gene expression of K133WT vs. K133AS-R parasites on the chromosome map. Chromosome map for differential gene expression was generated using Custom R program. Red lines indicate upregulated genes whereas green lines indicate downregulated genes in the K133AS-R parasite. (B) Percentage of differentially expressed genes in K133AS-R parasites. The percentage of modulated genes was calculated from the total 9170 genes obtained in Quality Control (QC) after filtering. Overall, 1.11% of genes were upregulated (red) whereas 1.15% of genes were downregulated (green); however, 97.74% of genes remained unaltered in K133AS-R parasites. (C) Categorization of genes showing differential expression in K133AS-R parasites according to GO functional categories. GO categories of differentially expressed genes in K133AS-R parasites suggested that genes belonging to various functional categories, such as metabolic processes, oxidation-reduction, cell membrane proteins, stress proteins, transporter activity, cell movement, and cell signaling, showed modulated expression. Unclassified proteins included hypothetical proteins with unknown function (that have not been characterized experimentally).

Figure 5

Comparative transcriptome profiling of K133WT and K133AS-R isolate. (A) Comparative gene expression of K133WT vs. K133AS-R parasites on the chromosome map. Chromosome map for differential gene expression was generated using Custom R program. Red lines indicate upregulated genes whereas green lines indicate downregulated genes in the K133AS-R parasite. (B) Percentage of differentially expressed genes in K133AS-R parasites. The percentage of modulated genes was calculated from the total 9170 genes obtained in Quality Control (QC) after filtering. Overall, 1.11% of genes were upregulated (red) whereas 1.15% of genes were downregulated (green); however, 97.74% of genes remained unaltered in K133AS-R parasites. (C) Categorization of genes showing differential expression in K133AS-R parasites according to GO functional categories. GO categories of differentially expressed genes in K133AS-R parasites suggested that genes belonging to various functional categories, such as metabolic processes, oxidation-reduction, cell membrane proteins, stress proteins, transporter activity, cell movement, and cell signaling, showed modulated expression. Unclassified proteins included hypothetical proteins with unknown function (that have not been characterized experimentally).

Figure 6

Validation of modulated expression of selected genes by qPCR. Selected 14 genes showing modulated expression in a microarray were validated for their altered expression by q-PCR in three independent RNA preparations. Fold changes in the gene expression of K133AS-R parasites with respect to K133WT parasites ± SD, obtained by q-PCR and microarray experiments, are represented here. The q-PCR data were normalized using two endogenous controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cystathionine β-synthase (CBS).

Figure 7

Expression analysis of AQP1 and HSP70 by Western blotting. Western blot analysis for the expression of AQP1 and HSP70 and α tubulin (endogenous control) protein was performed using 100 μgpromastigote cell lysates of K133WT and K133AS-R parasites. Proteins separated on a 12% SDS–PAGE gel, transferred to nitrocellulose membranes that were probed with anti-AQP1, anti-HSP70, or anti-α tubulin antibody followed by horseradish peroxidase (HRP)-conjugated antibody and developed using enhanced chemiluminescence (ECL).

Figure 8

Susceptibility of K133WT/K133AS-R isolates in the presence of the AQP1 inhibitor or the modulator to ABC transporter, verapamil. In vitro susceptibility of the sensitive wild-type strain K133 WT/artemisinin-resistant strain K133AS-R isolates towards artesunate in the presence of the AQP1 inhibitor (AQP1 Inh.) and verapamil (Vera) at (A) the promastigote stage and (B) amastigote stage. IC₅₀ ± SD of three independent experiments in duplicates is represented here. *** represents p ≤ 0.001, **** represents p ≤ 0.0001, NS represents not significant, Circle represents IC₅₀ of K133WT, Triangle represents IC₅₀ of K133AS-R.

Figure 9

Transcriptome predicted adaptations contributing artesunate resistance in L. donovani: Genes altered in K133AS-R parasites are represented here. Genes marked with an up and down arrow represent, respectively, the upregulated genes and the downregulated genes in K133AS-R parasites. 1, 2, 3, 4, 5, and 6 are probable adaptations in K133AS-R parasites. (1.) Downregulation of Atg8 and HSP70 leads to increased ROS production, which was compensated by upregulation in the expression of GSH1, (2.) Upregulated expression of enzymes involved in amino acid and lipid metabolism and downregulated expression of the enzyme involved in carbohydrate metabolism, suggesting a dependency on these metabolites for energy generation, (3.) Reduced DNA synthesis that leads to the parasites in the quiescence state may be responsible for artesunate resistance in Leishmania, (4.) Reduced protein synthesis and reduced protein degradation, (5.) Upregulated expression of AQP1 leads to higher nutrient uptake and increased discharge of waste material and metabolic end product from the parasites and (6.) Upregulated expression of ABC transporter (ABCG1) and partial reversion or resistance in the presence of the ABC transporter modulator verapamil suggested probable involvement of the ABC transporter in the efflux of artesunate drug.