Prevention and treatment of human malaria is heavily dependent upon the availability of effective antimalarial drugs, since there are no effective vaccines currently available. Unfortunately, the repertoire of antimalarial drugs effective against Plasmodium falciparum, the principal causative agent of human malaria, is dwindling due to widespread drug resistance. Gene products that perform an essential cellular function in the asexual blood stage have the greatest potential as targets for novel drugs. Reverse genetic techniques that are routinely used in other pathogenic organisms to verify essential genes either have been shown to not work, or are not yet compatible for use in the malaria parasite. Target verification, the initial required step in rational drug development, is greatly hampered without the proper genetic tools. Numerous presumptive targets for rational drug design may be proposed among the ~5,300 predicted gene sequences from the P. falciparum genome, but the currently available methods for identifying essential gene products in P. falciparum are inefficient and for the present purpose yield inconclusive negative results. New and robust methods employing alternative reverse genetic technologies are urgently needed to identify proteins required by the parasite to perform critical cellular functions. Recent reports of functional tetracycline (Tet)-regulated gene expression and high-efficiency transposon-based transformation in P. falciparum demonstrate that alternative approaches for genetic manipulation of the parasite are feasible. The objective of the proposed study is to define parameters of Tet-regulated gene expression in P. falciparum to develop an inducible gene-knockdown system that will make possible the positive identification of genes that are essential for the growth of the asexual, intraerythrocytic parasite.
Two specific aims are designed to pursue this objective:
Aim 1. Place the control of expression of a non-essential nuclear gene of P. falciparum under a Tet-regulated promoter to define suitable conditions for regulating its expression and assess the suitability of the system to knockdown expression of an essential gene.
Aim 2. Place expression of a nuclear gene, presumed to be essential, under control of the Tet-regulated promoter, conditionally knockdown that gene with the addition of anhydrotetracycline, and assess the knockdown phenotype. Sustaining our capacity to prevent and treat human malaria is dependent upon developing new, effective antimalarial drugs, since resistance to drugs now in use is widespread, and no effective vaccine is available. New antimalarial drugs may be rationally designed that inhibit the functioning of parasite gene products that perform essential cellular processes. The objective of this study is to develop genetic methods that will positively identify genes performing processes essential for growth of the malaria parasite, thus completing a critical first step toward drug development. ? ? ?
