Genomic study of Plasmodium falciparum gene regulation and mechanisms of drug action and resistance

Plasmodium falciparum, the causative agent of the deadliest type of human malaria continues to be a major cause of morbidity and mortality in many parts of the world. Because of the difficult nature of genetic and biochemical manipulations in P. falciparum genomic approaches are an increasingly important tool for studying this organism. We applied genomic approaches to learn about three distinct aspects of P. falciparum biology: mRNA decay during the intraerythrocytic developmental cycle (IDC), the antimalarial mechanism of tetracycline antibiotics, and the relationship between chloroquine resistance and mutations in the P. falciparum chloroquine resistance transporter (PfCRT). To study mRNA decay in P. falciparum, we used a genome-wide approach to characterize the decay profile of every gene in the genome during the IDC. We found that half-lives for all genes increased during the IDC, indicating that life-cycle is a major determinant of decay rate in P. falciparum. We also found that specific variations in decay patterns were superimposed upon the dominant trend of progressive half-life lengthening. These variations in decay pattern were frequently enriched for genes with specific cellular functions or processes, making gene function a secondary determinant of decay rate. Next, we used genomic techniques to study the antimalarial mechanism of action of tetracycline antibiotics. Using DNA microarrays to characterize the transcriptome over two generations after treatment with doxycycline, we found that expression of apicoplast genes is specifically inhibited, even if the drug was removed after the first generation. This inhibition is likely caused by a disruption of apicoplast function as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogony. Lastly, to study chloroquine resistance mediated by mutations in PfCRT we used Saccharomyces cerevisiae as a heterologous system to begin characterizing the structure-function relationship of PfCRT. We used DNA microarrays to find yeast genes which respond in a dose-dependent manner to chloroquine. These genes will be a starting point towards building a platform for characterizing resistance profiles of new 4-amino-quinoline drugs