Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription

Abstract

Background: Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates.

Results: In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype.

Conclusions: The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo.

Figure 1

Transcriptome analyses of the ex vivo cultured P. falciparum parasites. (a) Transcriptomes of the ex-vivo IDC of 6 field isolates (representative set of total 11 transcriptomes generated in this study; Additional file 3) from the 3 geographical locations over 48 hour sampling time. The heat maps represent the mean-centered log₂ microarray expression ratios for the P. falciparum genes that were ordered according to the phase calculated by the Fast Fourier Transformation for the reference in vitro IDC transcriptome. (b) Age estimate of the ex vivo culture time points for all 11 field isolates. Each colored box represents age estimate (hpi; shown by numbers outside the circle) of the isolate sample relative to the in vitro reference IDC transcriptome calculated as a best fit Correlation (Spearman rank, see materials and methods). Indicated within each box is the sampling collection time with respect to the first sampling time denoted by 0 h that correspond to the initial sample collection from the infected patients prior to culturing. Sampling times with * represent Spearman Rank correlation value less than 0.35 which indicates a deteriorating synchronicity in the later IDC time points and were excluded from the analysis. Time points included in 3 windows (yellow frame) corresponding to rings at 12-16 hpi, trophozoites at 24 to 28 hpi and schizonts at 32 to 36 hpi were selected for further analysis. (c) Clusters of genes with significant differential expression (p-value < 0.01) between the resistant (grey) and susceptible (orange) parasites at ring (14 hpi), trophozoite (26 hpi) and schizont (34 hpi) stages. Shown are the mean-centered expression log₂ ratios for these genes ranked by the z-score based on the differential expression between the resistant and susceptible isolates. Graphs represent the frequency distribution of the peak abundance time in the IDC transcriptome for each group of genes: over-expressed (red bars), under-expressed (green bars) or no significant change in expression (yellow bars). The grey lines represent the middle of the time IDC interval used for the analysis (e.g. ring, (14 hpi), trophozoite (26 hpi) and schizonts (34 hpi).

Figure 2

Functional analyses of differentially expressed genes in the artemisinin resistant parasites. (a) The heat maps depict differential gene expression in the trophozoite and schizont stages and the mean-centered log₂ ratios of mRNA levels between the resistant and susceptible isolates (for the complete set of all stages, see Additional file 6). Here the genes were ranked according to descending z-score (SNR) by correlating the expression profiles to the phenotypic class. Gene Set Enrichment Analysis (GSEA) revealed functional pathways down-regulated in trophozoites and up-regulated in schizonts of the resistant parasites shown in the side bar diagrams and ordered by the nominal p-value. Positions of the genes belonging to each identified pathway are indicated by the colored bars in the corresponding z-score ordered gene distribution. (b) Graphs illustrate several functional pathways with significant differential expression between the resistant and susceptible parasites in the three stages. Data points represent the average log₂ expression ratio for the isolates in each of the groups and across all genes belonging to the pathway for ring (14 hpi), trophozoite (26 hpi) and schizont (34 hpi) stages of the resistant (green triangle) and susceptible (blue diamond) isolates. Plotted are best fit polynomial curves and error bars that indicate the standard deviation amongst the isolates. Included are the average expression ratios for the artemisinin sensitive Cambodian isolate, CP022 (purple circle). For reference, the data are projected onto the centered mRNA abundance profiles of the in vitro IDC transcriptome (red square).

Figure 3

Classification of differentially expressed genes across the whole IDC and validation of differential expression of 5 genes by qPCR. (a) Plot of the Ranked Product Score for all 4,015 genes calculated from the geometric mean of the ranked z-scores of the genes in the 3 stages in the generated transcriptome (orange) and in the randomized dataset (green) (for details see material and methods). The graphs represent 4 over (b) and 4 under-expressed (c) genes in the resistant parasites with potential regulatory functions present in the top 5% of each extreme of the rank product distribution, respectively ((a) grey boxes). Data points represent the mean log₂ expression ratios of the genes in the resistant (green triangle), susceptible Lao and Thai (blue diamond), and susceptible Cambodian isolates (CP022, purple circle) across the three selected stage intervals. The data are projected onto the gene expression profiles analyzed by the in vitro IDC transcriptome (red square). Error bars reflect the standard deviation of the log₂ ratios for each data point. (d) Bars represent the log₂-transformed fold change measured from relative quantification of a resistant versus a sensitive isolate using PFC0965w as a reference control gene in real-time PCR experiments (teal) and plotted alongside the microarray expression ratios (light blue). Error bars reflect the standard deviation of the log₂ ratios over triplicates.

Figure 4

CGH analysis of artemisinin resistant isolates, Genotyping of isolates and Sequencing of drug-resistant genes to determine haplotypes. (a) The heat map represents hierarchical clustering of CGH signal (against 3d7 genome) for 257 microarray oligonucleotide elements representing 138 genes in 93 CNV regions identified by GADA analysis (see material and methods). (b) Visualization of msp1, msp2 and glurp nested PCR products of the 6 isolates on a 2.5% agarose gel shows distinctive patterns of product sizes for each clone, except for CP025 and CP037. (c) Tables depict the commonly and rarely found single nucleotide polymorphisms (SNPs) found based on the codon position (top row) and the sequenced bases of the various isolates and corresponding amino acid in brackets. Highlighted in yellow are the codons with mutations compared to wild type 3d7.