Moving “Forward” in Plasmodium Genetics through a Transposon-Based Approach

The genome sequence of the human malaria parasite, Plasmodium falciparum, was released almost a decade ago. A majority of the Plasmodium genome, however, remains annotated to code for hypothetical proteins with unknown functions. The introduction of forward genetics has provided novel means to gain a better understanding of gene functions and their associated phenotypes in Plasmodium. Even with certain limitations, the technique has already shown significant promise to increase our understanding of parasite biology needed for rationalized drug and vaccine design. Further improvements to the mutagenesis technique and the design of novel genetic screens should lead us to some exciting discoveries about the critical weaknesses of Plasmodium, and greatly aid in the development of new disease intervention strategies

A Genetic Screen for Attenuated Growth Identifies Genes Crucial for Intraerythrocytic Development of Plasmodium falciparum

A majority of the Plasmodium falciparum genome codes for genes with unknown functions, which presents a major challenge to understanding the parasite's biology. Large-scale functional analysis of the parasite genome is essential to pave the way for novel therapeutic intervention strategies against the disease and yet difficulties in genetic manipulation of this deadly human malaria parasite have been a major hindrance for functional analysis of its genome. Here, we used a forward functional genomic approach to study P. falciparum and identify genes important for optimal parasite development in the disease-causing, intraerythrocytic stages. We analyzed 123 piggyBac insertion mutants of P. falciparum for proliferation efficiency in the intraerythrocytic stages, in vitro. Almost 50% of the analyzed mutants showed significant reduction in proliferation efficiency, with 20% displaying severe defects. Functional categorization of genes in the severely attenuated mutants revealed significant enrichment for RNA binding proteins, suggesting the significance of post-transcriptional gene regulation in parasite development and emphasizing its importance as an antimalarial target. This study demonstrates the feasibility of much needed forward genetics approaches for P. falciparum to better characterize its genome and accelerate drug and vaccine development

piggyBac is an effective tool for functional analysis of the Plasmodium falciparum genome

<p>Abstract</p> <p>Background</p> <p>Much of the <it>Plasmodium falciparum </it>genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the <it>Plasmodium </it>genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the <it>Plasmodium </it>genome.</p> <p>Results</p> <p>In this study, we investigated the lepidopteran transposon, <it>piggyBac</it>, as a molecular genetic tool for functional characterization of the <it>Plasmodium falciparum </it>genome. Through multiple transfections, we generated 177 unique <it>P. falciparum </it>mutant clones with mostly single <it>piggyBac </it>insertions in their genomes. Analysis of <it>piggyBac </it>insertion sites revealed random insertions into the <it>P. falciparum </it>genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in <it>P. falciparum </it>with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development.</p> <p>Conclusion</p> <p>Our results clearly demonstrate that <it>piggyBac </it>is a novel, indispensable tool for forward functional genomics in <it>P. falciparum </it>that will help better understand parasite biology and accelerate drug and vaccine development.</p

The Malaria Secretome: From Algorithms to Essential Function in Blood Stage Infection

The malaria agent Plasmodium falciparum is predicted to export a “secretome” of several hundred proteins to remodel the host erythrocyte. Prediction of protein export is based on the presence of an ER-type signal sequence and a downstream Host-Targeting (HT) motif (which is similar to, but distinct from, the closely related Plasmodium Export Element [PEXEL]). Previous attempts to determine the entire secretome, using either the HT-motif or the PEXEL, have yielded large sets of proteins, which have not been comprehensively tested. We present here an expanded secretome that is optimized for both P. falciparum signal sequences and the HT-motif. From the most conservative of these three secretome predictions, we identify 11 proteins that are preserved across human- and rodent-infecting Plasmodium species. The conservation of these proteins likely indicates that they perform important functions in the interaction with and remodeling of the host erythrocyte important for all Plasmodium parasites. Using the piggyBac transposition system, we validate their export and find a positive prediction rate of ∼70%. Even for proteins identified by all secretomes, the positive prediction rate is not likely to exceed ∼75%. Attempted deletions of the genes encoding the conserved exported proteins were not successful, but additional functional analyses revealed the first conserved secretome function. This gave new insight into mechanisms for the assembly of the parasite-induced tubovesicular network needed for import of nutrients into the infected erythrocyte. Thus, genomic screens combined with functional assays provide unexpected and fundamental insights into host remodeling by this major human pathogen

The Transmembrane Isoform of Plasmodium falciparum MAEBL Is Essential for the Invasion of Anopheles Salivary Glands

Malaria transmission depends on infective stages in the mosquito salivary glands. Plasmodium sporozoites that mature in midgut oocysts must traverse the hemocoel and invade the mosquito salivary glands in a process thought to be mediated by parasite ligands. MAEBL, a homologue of the transmembrane EBP ligands essential in merozoite invasion, is expressed abundantly in midgut sporozoites. Alternative splicing generates different MAEBL isoforms and so it is unclear what form is functionally essential. To identify the MAEBL isoform required for P. falciparum (NF54) sporozoite invasion of salivary glands, we created knockout and allelic replacements each carrying CDS of a single MAEBL isoform. Only the transmembrane form of MAEBL is essential and is the first P. falciparum ligand validated as essential for invasion of Anopheles salivary glands. MAEBL is the first P. falciparum ligand experimentally determined to be essential for this important step in the life cycle where the vector becomes infectious for transmitting sporozoites to people. With an increasing emphasis on advancing vector-based transgenic methods for suppression of malaria, it is important that this type of study, using modern molecular genetic tools, is done with the agent of the human disease. Understanding what P. falciparum sporozoite ligands are critical for mosquito transmission will help validate targets for vector-based transmission-blocking strategies

A highly sensitive, PCR-based method for the detection of <it>Plasmodium falciparum </it>clones in microtiter plates

<p>Abstract</p> <p>Background</p> <p>Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is time-consuming, whereas the more sensitive parasite lactate dehydrogenase assay involves multiple steps and requires fresh reagents. A simple PCR-based method was therefore tested for parasite detection that can be adapted to high throughput studies.</p> <p>Methods</p> <p>Approximately 1 μL of packed erythrocytes from each well of a microtiter cloning plate was directly used as template DNA for a PCR reaction with primers for the parasite <it>18s rRNA </it>gene. Positive wells containing parasites were identified after rapid separation of PCR products by gel electrophoresis.</p> <p>Results</p> <p>The PCR-based method can consistently detect a parasitaemia as low as 0.0005%, which is equivalent to 30 parasite genomes in a single well of a 96-well plate. Parasite clones were easily detected from cloning plates using this method and a comparison of PCR results with Giemsa-stained blood smears showed that PCR not only detected all the positive wells identified in smears, but also detected wells not identified otherwise, thereby confirming its sensitivity.</p> <p>Conclusion</p> <p>The PCR-based method reported here is a simple, sensitive and efficient method for detecting parasite clones in culture. This method requires very little manual labor and can be completely automated for high throughput studies. The method is sensitive enough to detect parasites a week before they can be seen in Giemsa smears and is highly effective in identifying slow growing parasite clones.</p