Exploring the Plasmodium falciparum Transcriptome Using Hypergeometric Analysis of Time Series (HATS)

Malaria poses a significant public health and economic threat in many regions of the world, disproportionately affecting children in sub-Saharan Africa under the age of five. Though success has been celebrated in lowering infection rates, it remains a serious challenge, causing at least 200 million infections and 655,000 deaths per year, with deleterious effects on economic growth and development. Investigation of the malaria parasite Plasmodium falciparum has entered the post-genomics age, with several strains sequenced and many microarray gene expression studies performed. Gene expression studies allow a full sampling of the genomic repertoire of a parasite, and their detailed analysis will prove invaluable in deciphering novel parasite biology as well as the modes of action of antimalarial drug resistance. We have developed a computational pipeline that converts a series of fluorescence readings from a DNA microarray into a meaningful set of biological hypotheses based on the comparison of two lines, generally one that is drug sensitive and one that is drug resistant. Each step of the computational pipeline is described in detail in this thesis, beginning with data normalization and alignment, followed by visualization through dimensionality reduction, and finally a direct analysis of the differences and similarities between the two lines. Comparisons and analyses were performed at both the individual gene and gene set level. An important component of the analytical methods we have developed is a suite of visualization tools that help to easily identify outliers and experimental flaws, measure the significance of predictions, show how lines relate and how well they can be aligned, and demonstrate the results of an analysis. These visualization tools should be used as a starting point for further biological study to test the resulting hypotheses. We also developed a software tool, Gene Attribute and Set Enrichment Ranking (GASER), which combines a wealth of genomic data from the TDR Targets web site along with expression data from a variety of sources, and allows researchers to create sophisticated weighted queries to undercover potential drug targets. Queries in our system can be updated in real time, along with their accompanying gene and gene set lists. We analyzed all possible pair-wise combinations of 11 parasite lines to create baseline distributions for gene and gene set enrichment. Using the baseline as a comparison, we identified and discarded spurious results and recognized stochastic genes and gene sets. We analyzed three major sets of parasite lines: those involving manipulation of the multidrug resistance-1 (PfMDR1) transporter, a key resistance determinant; those involving manipulation of the P. falciparum chloroquine resistance transporter (PfCRT), another important resistance determinant; and finally a set of parasites that had varying sensitivity to artemisinins. This analysis resulted in a rich library of high scoring genes that may merit further exploration as potential modes of action of resistance. More specifically, we found that manipulation of pfcrt expression resulted in an up-regulation of tRNA synthetases, which might serve to increase protein production in response to reduced amino acid availability from degraded hemoglobin. We observed that a copy number increase in pfmdr1 resulted in increases in glycerophospholipid metabolism and up-regulation of a number of ABC transporters. Finally, when comparing artemisinin sensitive to artemisinin tolerant lines, we found an increased abundance of redox metabolites and the transcripts involved in redox regulation, and significant reduction in transcription and altered expression of transcripts encoding for core histone proteins. These alterations could help confer an increased tolerance to drug induced redox perturbation by lowering endogenous redox stress. We also offer a robust computational tool, Hypergeometric Analysis of Time Series (HATS), to handle challenging biological questions related to comparison of time series experiments. Our pipeline provides a rigorous method for aligning expression experiments and then determining which genes and gene sets differ most between them. The changes in gene expression level between drug-sensitive and drug-resistant lines offer important clues in our quest for understanding mechanisms of resistance and identifying new drug targets. Our pipeline allows for comparison of future lines with our base set and holds potential for other organisms, especially those similar to Plasmodium with a strong time-dependent component. The full excel files of all the analyses performed in this thesis can be found at: (http://www.fidock.org/dan)

Genome-wide transcriptome profiling reveals functional networks involving the Plasmodium falciparum drug resistance transporters PfCRT and PfMDR1

Background The acquisition of multidrug resistance by Plasmodium falciparum underscores the need to understand the underlying molecular mechanisms so as to counter their impact on malaria control. For the many antimalarials whose mode of action relates to inhibition of heme detoxification inside infected erythrocytes, the digestive vacuole transporters PfCRT and PfMDR1 constitute primary resistance determinants. Results Using gene expression microarrays over the course of the parasite intra-erythrocytic developmental cycle, we compared the transcriptomic profiles between P. falciparum strains displaying mutant or wild-type pfcrt or varying in pfcrt or pfmdr1 expression levels. To account for differences in the time of sampling, we developed a computational method termed Hypergeometric Analysis of Time Series, which combines Fast Fourier Transform with a modified Gene Set Enrichment Analysis. Our analysis revealed coordinated changes in genes involved in protein catabolism, translation initiation and DNA/RNA metabolism. We also observed differential expression of genes with a role in transport or coding for components of the digestive vacuole. Interestingly, a global comparison of all profiled transcriptomes uncovered a tight correlation between the transcript levels of pfcrt and pfmdr1, extending to dozens of other genes, suggesting an intricate regulatory balance in order to maintain optimal physiological processes. Conclusions This study provides insight into the mechanisms by which P. falciparum adjusts to the acquisition of mutations or gene amplification in key transporter loci that mediate drug resistance. Our results implicate several biological pathways that may be differentially regulated to compensate for impaired transporter function and alterations in parasite vacuole physiology

Investigations into the Role of the Plasmodium falciparum SERCA (PfATP6) L263E Mutation in Artemisinin Action and Resistance ▿ †

Artemisinin-based combination therapies (ACTs) are highly effective for the treatment of Plasmodium falciparum malaria, yet their sustained efficacy is threatened by the potential spread of parasite resistance. Recent studies have provided evidence that artemisinins can inhibit the function of PfATP6, the P. falciparum ortholog of the ER calcium pump SERCA, when expressed in Xenopus laevis oocytes. Inhibition was significantly reduced in an L263E variant, which introduced the mammalian residue into a putative drug-binding pocket. To test the hypothesis that this single mutation could decrease P. falciparum susceptibility to artemisinins, we implemented an allelic-exchange strategy to replace the wild-type pfatp6 allele by a variant allele encoding L263E. Transfected P. falciparum clones were screened by PCR analysis for disruption of the endogenous locus and introduction of the mutant L263E allele under the transcriptional control of a calmodulin promoter. Expression of the mutant allele was demonstrated by reverse transcriptase (RT) PCR and verified by sequence analysis. Parasite clones expressing wild-type or L263E variant PfATP6 showed no significant difference in 50% inhibitory concentrations (IC50s) for artemisinin or its derivatives dihydroartemisinin and artesunate. Nonetheless, hierarchical clustering analysis revealed a trend toward reduced susceptibility that neared significance (artemisinin, P ≈ 0.1; dihydroartemisinin, P = 0.053 and P = 0.085; and artesunate, P = 0.082 and P = 0.162 for the D10 and 7G8 lines, respectively). Notable differences in the distribution of normalized IC50s provided evidence of decreased responsiveness to artemisinin and dihydroartemisinin (P = 0.02 for the D10 and 7G8 lines), but not to artesunate in parasites expressing mutant PfATP6

Additional file 22: Figure S8. of Genome-wide transcriptome profiling reveals functional networks involving the Plasmodium falciparum drug resistance transporters PfCRT and PfMDR1

Cluster heatmap of gene expression data for pfcrt/pfmdr1 and all other genes. The hierarchical clustering was generated using PCC values calculated using log2-transformed and normalized expression values of 2,600 genes across 110 pairwise comparisons of 11 parasite transcriptome data sets, corresponding to all possible combinations in both directions (i.e. A vs. B and B vs. A). Squares indicate areas containing genes that are all strongly correlated or strongly anti-correlated with the expression of both pfcrt and pfmdr1. (PDF 499 kb

Additional File 25:

Zipped file folder containing code and pseudocode for HATS analysis. These files can be downloaded from http://www.fidock.org/data/fidock_source_code.zip . (DOCX 32 kb

Additional File 26: Table S17.

Table summarizing all the gene sets, extracted from various databases or built by our group, that were used in this study. (XLSX 688 kb

Additional file 17: Table S11. of Genome-wide transcriptome profiling reveals functional networks involving the Plasmodium falciparum drug resistance transporters PfCRT and PfMDR1

Genes with significant differential expression between 7G8 and 7G8 pfcrt_T76K in a stage-specific manner. Genes with significantly higher expression displayed absolute fold changes >1.5 and normalized fold changes values two standard deviations above the median normalized fold change value. Genes with significantly lower expression had absolute fold change values lower than 0.6 and a normalized fold change three standard deviations below the median value. Fold change for each gene at the designated stage is indicated. (XLSX 59 kb

Additional file 8: Table S4. of Genome-wide transcriptome profiling reveals functional networks involving the Plasmodium falciparum drug resistance transporters PfCRT and PfMDR1

Values for gene expression fold differences between strains in the 7G8 data set for genes on chromosome 2. The boxed area corresponds to the region on chromosome 2 containing genes with a significant fold change (values <0.6). (XLSX 13 kb