Use of variability in the stage-specific transcription levels of Plasmodium falciparum in the selection of target genes

Abstract

The malarial parasite Plasmodium falciparum exhibits several morphological and developmental stages. We have quantified the level of expression of a battery of genes in the ring and trophozoite stage-two of the most prominent stages in the erythrocytic development of the parasite. Using optimized RT-PCR, we observed that some of the genes show a large variation in stage-specific expression. We have also correlated the level of mRNA expression (of the target enzyme) to its metabolic requirement using specific inhibitors. This protocol gives us a handle to identify vulnerable target genes that could be used to develop antimalarials.

Introduction

The malarial parasite Plasmodium falciparum goes through a complex set of developmental growth patterns during its passage from mosquito to man. Its growth pattern is generally optimized to the corresponding host cell that it infects. Even in simplified in vitro culture, which parallels the erythrocytic stage of growth, it passes through stages described as rings, trophozoites and schizonts. As these stages have different morphological characteristics, their metabolic and expression profiles would seem to be different. There is evidence of strong stage-specific expression of genes [1]. In a recent report, a set of genes that are exclusively transcribed in the ring stage of the parasite have been identified [2]. While screening for antimalarial compounds in in vitro culture, not much attention has been paid to the stages of parasite growth. Unless specialized techniques are adopted, the parasites in in vitro culture generally exhibit a randomized mixture of all stages of growth [3]. In contrast, in P. falciparum-infected humans, the parasites show a synchrony in their growth pattern [4]. Recent studies indicate that antimalarials in general show variability in the inhibition pattern (IC₅₀), depending on the stage of growth [5]. Drug targets in pathogens are specific enzymes involved in an essential metabolic pathway with no bypass. If antimalarials act as enzyme inhibitors, then it is logical to assume that the stage-specific action of drugs is the result of stage-specific expression levels of the target enzyme. Inhibition of growth due to enzyme inhibition is either due to a toxic build-up of the substrate of the enzyme being inhibited, or to a reduction in the metabolic flux (of the enzymatic pathway) to below survival levels. The mathematical modeling of an in vivo enzyme situation using metabolic control analysis (MCA) has shown that the correlation between enzyme inhibition and either substrate build-up or reduction of metabolic flux is non-linear [6]. Hence, IC₅₀ of parasite growth is not generally equivalent to a 50% in vivo inhibition of a target enzyme. A good drug target would only require moderate inhibition to produce a large growth inhibition. It is thus critical that when rational drug design is attempted on the basis of inhibition of a certain key enzyme, the relative level of the enzyme in various stages of growth be monitored. Inhibition of the target enzyme can then be correlated to its expression level and the inhibition of the growth of the parasite.

Although there are some exceptions [7], it is a generally accepted concept that the level of polyadenylated mRNA corresponds to the relative level of its translated product. We have used this concept to monitor the stage-specific expression by RT-PCR of a battery of key enzymes in the parasite P. falciparum and have attempted to correlate the growth inhibition by specific enzyme inhibitors to the expression level of the gene.