Dissecting Mechanisms of Antimalarials using CRISPR/Cas9 Editing in Plasmodium falciparum

Thesis (Ph.D.)--University of Washington, 2019Malaria, caused by Plasmodium infections, continues to be a global disease of public health importance with 300 million annual cases and about 500,000 deaths. Continual emergence of resistance to commonly used antimalarials underscores the importance of finding new drug targets and new antimalarial drugs. Previously, the Rathod lab has established systematic approaches to study targets of antimalarials and resistance mechanisms with the use of in vitro selection methods and deep sequencing of selected mutants. There are some limitations with these approaches as deep sequencing data does not reveal the stepwise mechanism of mutagenesis and mutations observed from the sequencing result might not associate with the resistance phenotype. This thesis has multiple projects aimed to expand the molecular toolbox with genome manipulation using CRISPR/Cas9 technique. It will complement the current tools that we have in performing target identification/validation as well as understanding the mechanism of mutagenesis in malaria parasites. Ciprofloxacin is an antibacterial known to target bacterial DNA Gyrase. In some instances, ciprofloxacin has been used for malaria prophylaxis but little is known about the mode-of-action of ciprofloxacin in malaria parasites. In the first project, we aim to understand the essentiality of Plasmodium falciparum DNA gyrase A subunit (PfGyrA) and its relationship with ciprofloxacin. Based on bioinformatics analyses, PfGyr A and B subunits are known to contain apicoplast-targeting signals. To test the predicted localization of this enzyme in the apicoplast and the function of this enzyme at the subcellular level, a CRISPR/Cas9 gene-editing tool was used to disrupt PfGyrA. It is known that isopentenyl pyrophosphate (IPP) rescues malaria parasites from apicoplast-targeting inhibitors and indeed successful growth of PfΔGyrA required chemical rescue with IPP. PfGyrA disruption was accompanied by loss of the plastid acyl-carrier protein (ACP) immunouorescnce and the plastid genome. Drug sensitivity assays revealed that a PfΔGyrA clone, supplemented with IPP was less sensitive to antibacterial compounds (doxycycline and ciprofloxacin) but not the nuclear topoisomerase inhibitor (etoposide). In addition, at high concentrations, ciprofloxacin continued to inhibit IPP-rescued PfΔGyrA suggesting that this drug has an additional target in P. falciparum. We concluded that PfGyrA is an apicoplast enzyme in malaria parasite and it is essential for blood-stage parasites. In the future, untangling the two possible inhibitory functions of ciprofloxacin in malaria parasites may reveal a new and important drug target. The second project aim involves target validation of a tetrahydroquinolone compound, BMS-388891. Previous publications from the lab showed that resistance to BMS-388891 arises from a single point mutation in either the protein farnesyl transferase (PFT) alpha or beta subunit. Although results indicated that a single point mutation on the PfPFT enzyme led to BMS-3888891 resistant parasites, whole genome sequencing on those mutants have yet to be done. To test that a single mutation is sufficient for parasite acquisition of resistance to BMS-388891, gene alteration with CRISPR/Cas9 tool was utilized to introduce a point mutation (Y837N, Y837S, or Y837C) on the PFT-β-subunit. The CRISPR-modified mutant parasites have shown an increase of 10-20 fold resistance to BMS-388891. This data is the first to formally demonstrate that a single point mutation on the Pfpft-β-subunit is sufficient for parasites to confer resistance to BMS-388891 compound. There are very few validated compound to target relationships and CRISPR/Cas9 technique will be a valuable tool in the malaria field. The third project aim involves the understanding of the mechanism of mutagenesis in malaria parasites. While it is known that amplification and point mutation are the possible outcomes of resistance selection, the order of the processes is less understood. Recent work by Guler et. al. points to a novel step-wise amplification mechanism in the malarial parasite response to DSM1 selection pressure. In these selected parasites, 25-30 kb regions surrounding the Pfdhodh locus were amplified. Taking advantage of the highly amplified Pfdhodh locus, we were able to introduce Pfpft-α-subunit into this region. This sets up future studies for us to dissect the step-wise resistance mechanism in malaria parasites. Overall, the utilization of CRISPR/Cas9 tool has allowed us to efficiently perform gene knockout, gene alteration and gene translocation. These applications not only enable us to prove for the first time the importance of the PfGyrA enzyme but also to directly confirm the causality of specific point mutations in BMS-388891 resistant parasites. The addition of CRISPR/Cas9 gene-editing to our systematic approach toolbox will ultimately aid in our understanding of how mutagenesis occurs in malaria parasites and allow us to expand our knowledge in the mode-of-action of different antimalarials in P. falciparum