Expression and genomic changes after exposing drug-selected mutants to short term CQ treatment in Plasmodium falciparum.. Plasmodium falciparum

Mutations in PfCRT confer chloroquine (CQ) resistance in P. falciparum. Point mutations in the homolog of the mammalian multidrug resistance gene (pfmdr1) can also modulate the levels of CQ response. However, parasites with the same pfcrt and pfmdr1 alleles exhibit a wide range of drug sensitivity, suggesting that additional genes contribute to levels of CQ resistance (CQR). We used 3 isogenic lines which have different drug resistance profiles corresponding to unique mutations in the pfcrt gene (106/1K76, 106/176I, and 106/76I-352K) to study changes in gene expression with and without CQ and genomic variations, i.e. copy number (CN) changes. RNA transcription levels from 45 genes were significantly altered in one or both mutants relative to the parent line. Of particular interest are genes encoding proteins involved in transport and/or regulation of cytoplasmic or compartmental pH, e.g. the V-type H+ pumping pyrophosphatase 2, Ca2+/H+ antiporter VCX1 and copy number changes in pfmdr1. A series of deletion (including 15 genes) also occurred at the beginning of chromosome 10. Keywords: low-dose 3h Chloroquine response, copy number variation Overall design: Three isogenic parasite lines were treated with and without 3h CQ and synchronized for total RNA extraction and hybridization to Affymetrix GeneChips® to study gene expression profiles. Genomic DNA from mixed culture was also extracted and hybridized to the same Affymetrix GeneChips. Total 18 total RNA samples (3 biological replicates per condition) and 6 genomic DNA samples (2 biological replicates per condition).