EVALUATION OF A SURROGATE IN VITRO MAMMALIAN LOCALIZATION SYSTEM FOR RECOGNITION OF PLASMODIAL FOOD VACUOLE TARGETING SIGNAL

The completion of Plasmodium falciparum genomic sequences had revealed approximately 5300 potential gene products with 60% annotated as hypothetical proteins. The main task is to define the functional roles of these putative proteins experimentally. The use of protein localization study could aid in this cause as the strategic location of cellular proteins underscores their functionality in living systems. This would help in resolving unknown metabolic pathways which are fundamentally important in understanding and controlling the parasite pathogenicity. A previous study had successfully employed an in vitro surrogate mammalian host system to evaluate plasmodial mitochondrialand nucleartargeted signals. In this study, the surrogate host system’s ability to recognize plasmodial food vacuole proteins was evaluated. Two protein domains carrying food vacuole signal peptides were made and subjected to co-localization study with organelle selective probe, LysoTracker Red. This fluorescent acidotropic probe is able to specificity stain acidic lysosomal organelles. The preliminary results had demonstrated that the surrogate host could recognize plasmodial food vacuole-targeting signal and deliver it to vesicle-like compartments. After which, co-localization studies identified these compartments as lysosomal organelles. This demonstrated that the authenticity of putative food vacuole proteins could be preliminary verified via a surrogate host system. INTRODUCTION The genome sequence of P. falciparum, a causative agent of malaria, was uncovered in year 2002 (Gardner et al. 2002). It revealed that an approximate of 5300 protein encoding genes was reported with 60% being hypothetical proteins with no similar homologs in other organisms to provide clues in their functionality (Gardner et al. 2002). These putative proteins hold the key in resolving remaining unknown metabolic pathways in the parasite. Characterizing and authenticating them would be fundamentally important for our understanding and controlling the parasite pathogenicity. Much research effort had been carried out to study P. falciparum genes. One of effort made was the design of in vitro mammalian cell as surrogate host to evaluate plasmodial protein localization (Chan et al, 2006). In that study, mammalian cells were able to translocate the plasmodial mitochondrialand nuclear-targeted signals to the respective organelles, thereby verifying that the cells are able to interpret plasmodial mitochondrialand nuclear-targeted signals. This experimental system offers a new and easily accessible alternative which is able to circumvent the technical glitch in transfecting parasites. By broadening the scope of using mammalian surrogate system to analyze other plasmodial organelle targeted putative proteins; it would help in defining the thousands of proteins that have been identified through genomics efforts. One of the utmost important organelles to investigate would be the malarial food vacuole. It is known that several biologically essential processes such as hemoglobin degradation, heme polymerization and reduction of oxidative stress occur in the food vacuole (Olliaro and Goldberg, 1996).