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Gene Expression Analysis

Gene expression studies promise to reveal changes in gene expression between the different stages of the parasite life cycle. Quantification of gene expression in Plasmodium spp. is an essential requirement for a full understanding of parasite biology and has been achieved at the level of transcription using SAGE [119, 120], quantitative real-time PCR [121-123], and with microarrays [124-126]. Microar-rays allow analysis of gene expression changes under a variety of conditions, or as a function of developmental stage. The Malaria Research and Reference Reagent Resource Center (MR4, www.malaria.mr4.org) has produced P.falciparum micro-arrays containing 70-nucleotide-long oligomers, representing the complete set of genes. To date, microarray studies have revealed an unusual program for transcription in the bloodstage cycle of the parasite, whereby each gene appears to go through a single episode of induction during the 48-h cycle, with many genes of related function being expressed at a similar point in a tightly regulated program [124-128]. Efforts to perturb this system via environmental stress or drug pressure may give an insight into the degree that asexual gene expression is "hard wired." Gene expression studies further helped to identify over 1300 genes transcribed in the sporozoite stage. Sporozoites are the liver-infective stage of the Plasmodium pathogen and, until recently, little was known about protein expression and antigen exposure in these forms. Recent studies identified a variety of novel genes that might code for potentially important surface molecules and proteins essential for the development of exoerythrocytic liver forms [129-131].

19.7.2 Proteomics

Proteomics refers to the large-scale analysis of proteins expressed under described conditions, within specific cellular compartments, or at a particular time in an organism's life cycle. Advances in proteomics will be crucial in identifying differentially expressed proteins, which along with comparative genomic analysis, utilization of protein interaction maps, and an understanding of metabolic pathways, will help identify and prioritize targets for therapeutic intervention. Traditional methods of characterizing and identifying large numbers of proteins from complex protein mixtures have relied predominantly on two-dimensional gel electro-phoresis [132] combined with N-terminal sequencing or mass spectrometry of individually prepared proteins. New proteomics methods are now available that are based upon resolving small peptides derived from complex protein mixtures by high-resolution liquid chromatography and directly identifying them by tandem

19.7 Postgenomic Strategies and the Search for Cure | 435

mass spectrometry [133, 134], followed by sophisticated computer search algorithms against whole-genome sequence databases. One of the powerful features of this approach to protein identification is the ability to identify proteins expressed associated with membrane fractions. These newer proteomics methods have the potential to promote the identification of large numbers of proteins from various life cycle stages of Plasmodium, in turn supporting a better understanding of parasite biology and leading to the identification of new vaccine and drug targets [135-137].

With the advent of whole-genome sequence data, two different approaches to investigating the P. falciparum proteome were made in 2002 [47, 138]. Lasonder and coworkers [47] identified 1289 proteins by high-accuracy mass spectrometric proteome analysis of P. falciparum blood stages, of which 714 proteins were identified in asexual blood stages, 931 in gametocytes, and 645 in gametes. In the second study, Florens et al. [138] investigated four stages of the parasite life cycle (sporozoites, merozoites, trophozoites, and gametocytes) using multidimensional protein identification methods and identified more than 2400 proteins. Interestingly, the antigenic variant proteins var and rif, previously defined as molecules on the surface of infected erythrocytes (see Section 19.4.1 and Fig. 19.2), were also largely expressed in sporozoites. The sporozoite proteome appeared markedly different from all other stages, and almost half of the sporozoite proteins were unique to this stage. In contrast to sporozoites, which share an average of 25% of proteins with any other stage, trophozoites, merozoites, and gametocytes had between 20% and 33% unique proteins and shared between 39% and 56% of their proteins. Consequently, only 6% of proteins are common to all four stages, and these were predominantly identified as housekeeping proteins.

The lifecycle-stage specificity of Plasmodium protein expression suggests that there is a highly coordinated expression of genes involved in common processes. Analysis of coregulated gene groups facilitates both searching for regulatory motifs common to upregulated genes, and predicting protein functions on the basis of the "guilt by association" model. When detected proteins were mapped onto all 14 chromosomes, a total of 98 clusters containing 3 loci, 32 clusters containing 4 loci, and 5 clusters containing 6 loci were identified [138]. The focus on analyses describing stage-specific multistage clusters will facilitate identifying stage-specific and general cis-acting sequences, and will help decipher gene expression regulation during the Plasmodium parasite life cycle.

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