The Plasmodium falciparum sexual development transcriptome: A microarray analysis using ontology-based pattern identification

Abstract

The sexual stages of malarial parasites are essential for the mosquito transmission of the disease and therefore are the focus of transmission-blocking drug and vaccine development. In order to better understand genes important to the sexual development process, the transcriptomes of high-purity stage I–V Plasmodium falciparum gametocytes were comprehensively profiled using a full-genome high-density oligonucleotide microarray. The interpretation of this transcriptional data was aided by applying a novel knowledge-based data-mining algorithm termed ontology-based pattern identification (OPI) using current information regarding known sexual stage genes as a guide. This analysis resulted in the identification of a sexual development cluster containing 246 genes, of which ∼75% were hypothetical, exhibiting highly-correlated, gametocyte-specific expression patterns. Inspection of the upstream promoter regions of these 246 genes revealed putative cis-regulatory elements for sexual development transcriptional control mechanisms. Furthermore, OPI analysis was extended using current annotations provided by the Gene Ontology Consortium to identify 380 statistically significant clusters containing genes with expression patterns characteristic of various biological processes, cellular components, and molecular functions. Collectively, these results, available as part of a web-accessible OPI database (http://carrier.gnf.org/publications/Gametocyte), shed light on the components of molecular mechanisms underlying parasite sexual development and other areas of malarial parasite biology.

Introduction

Malaria continues to be a devastating infectious disease, responsible for approximately 500 million clinical episodes and millions of deaths each year worldwide [1]. Development of drugs and vaccines to combat the disease traditionally has focused heavily on the intraerythrocytic stages of the malarial parasite life cycle, as these stages are responsible for the clinical symptoms associated with the illness. However, in recent decades, it has become apparent that any successful strategy for controlling malaria will most likely require a multifaceted approach that also includes drugs and vaccines against other stages of the complex parasite life cycle. Consequently, the sexual stages of the parasite essential for the mosquito transmission of the disease are considered attractive targets for the development of new transmission-blocking drugs and vaccines that aim to prevent the spread of malaria in human populations [2], [3]. Such strategies are thought to be especially promising, as it has been hypothesized that the parasite may be more vulnerable to vaccine and drug intervention during the sexual part of its life cycle due to its passage through a numerical bottleneck and the limited exposure to the human immune system it receives during these stages [3].

Of the four Plasmodium parasite species responsible for malaria in humans, the progression of sexual development is best understood morphologically for the most lethal species, Plasmodium falciparum [4], [5]. The switch from an asexual to sexual mode of replication begins in the haploid intraerythrocytic stages, where a sub-population of asexual parasites begin to develop into male and female gametocytes. This process of gametocyte development continues in the human host over a period of approximately 10 days, encompassing five morphologically defined gametocyte stages (stage I–V) and ending with the formation of mature male and female gametocytes. When mature gametocytes are taken up by a mosquito as part of the bloodmeal the process of sexual development continues in the mosquito midgut, where male gametocytes undergo exflagellation to form highly motile male gametes and female gametocytes enlarge and emerge from red blood cells to form female gametes. The subsequent fusion of a single male and female gamete results in fertilization and the formation of a diploid zygote that then differentiates into a motile ookinete. It is this ookinete stage that transverses the mosquito midgut wall to form an oocyst, where asexual sporogonic development is once again initiated.

Despite this detailed morphological description, comprehensive understandings of most of the fundamental biological mechanisms driving the parasite sexual development process remain elusive. For example, we still do not fully understand how gene and protein expression is regulated during sexual development and what specific metabolic differences exist between the asexual and sexual stages. As a result, current transmission-blocking vaccine development has focused on only a handful of known sexual-specific proteins, and chemotherapeutics that selectively kill sexual stage parasites have yet to be discovered [2], [3].

The availability of the full genome sequence for P. falciparum since 2002 has allowed for the investigation of gene functions in the malarial parasite using high-throughput genomic and proteomic approaches that circumvent some obstacles associated with the application of more traditional genetic and biochemical methods to malaria research [6], [7], [8]. Previously, we conducted a genome-wide transcriptional analysis of various stages of the P. falciparum life cycle using a high-density oligonucleotide microarray coupled with a k-means clustering approach to identify 15 groups of genes sharing mRNA expression patterns characteristic of various biological processes such as antigenic variation and cell invasion [9]. In an analogous study, Bozdech and co-workers utilized a spotted oligonucleotide array and Fourier transformation analysis to demonstrate that ∼80% of the P. falciparum genes expressed during the asexual intraerythrocytic cell cycle exhibit a single-peak periodic pattern in their transcript levels, with genes involved in common biological processes also sharing similar phase [10]. Together, these studies provided many novel insights into potential functions for the approximately 3000 hypothetical genes in the P. falciparum genome. However, due to the inclusion of only a single late-stage gametocyte time point in the former work, insights provided by these studies regarding sexual stage gene function were limited.

To better understand genes involved in P. falciparum sexual development using an expression microarray approach, we have obtained new transcriptional data on detailed time courses of gametocyte development, including high-purity early-stage gametocytes. To aid in the interpretation of this new microarray data within the context of our previous asexual stage data [9], we applied a recently developed knowledge-based clustering algorithm called ontology-based pattern identification (OPI) [11]. OPI utilizes classifications of gene function in the form of systematic gene annotations to generate gene clusters with greater specificity and statistical confidence than is possible using more routinely-used clustering methods such as the k-means clustering approach employed in the past [7], [9], [10]. For example, hierarchical or k-means methods require the user to arbitrarily select a correlation coefficient, a fold-change, or a number of k-clusters to determine cluster sizes. In contrast, OPI empirically selects the best parameters to give the highest concentration of genes belonging to a particular classification in the smallest cluster size. Using manually-assigned gene annotations for sexual development to guide OPI, a cluster of 246 genes possessing highly gametocyte-specific mRNA expression patterns was identified. Furthermore, inspections of promoter regions upstream of these genes revealed conserved sequences that potentially play roles as cis-regulatory elements in sexual development transcriptional control mechanisms. Beyond sexual development, OPI was also extended to identify genes most likely to be involved in 380 biological processes, cellular components, and molecular functions using information provided by the Gene Ontology (GO) Consortium [12]. Collectively, these 381 clusters, accessible as part of an OPI database at http://carrier.gnf.org/publications/Gametocyte, provide new insights into P. falciparum gene function with direct implications for rational drug and vaccine development against malaria.