Supergenomic network compression and the discovery of EXP1 as a glutathione transferase inhibited by artesunate.

A central problem in biology is to identify gene function. One approach is to infer function in large supergenomic networks of interactions and ancestral relationships among genes; however, their analysis can be computationally prohibitive. We show here that these biological networks are compressible. They can be shrunk dramatically by eliminating redundant evolutionary relationships, and this process is efficient because in these networks the number of compressible elements rises linearly rather than exponentially as in other complex networks. Compression enables global network analysis to computationally harness hundreds of interconnected genomes and to produce functional predictions. As a demonstration, we show that the essential, but functionally uncharacterized Plasmodium falciparum antigen EXP1 is a membrane glutathione S-transferase. EXP1 efficiently degrades cytotoxic hematin, is potently inhibited by artesunate, and is associated with artesunate metabolism and susceptibility in drug-pressured malaria parasites. These data implicate EXP1 in the mode of action of a frontline antimalarial drug.

PfSR1 controls alternative splicing and steady-state RNA levels in Plasmodium falciparum through preferential recognition of specific RNA motifs.

Plasmodium species have evolved complex biology to adapt to different hosts and changing environments throughout their life cycle. Remarkably, these adaptations are achieved by a relatively small genome. One way by which the parasite expands its proteome is through alternative splicing (AS). We recently identified PfSR1 as a bona fide Ser/Arg-rich (SR) protein that shuttles between the nucleus and cytoplasm and regulates AS in Plasmodium falciparum. Here we show that PfSR1 is localized adjacent to the Nuclear Pore Complex (NPC) clusters in the nucleus of early stage parasites. To identify the endogenous RNA targets of PfSR1, we adapted an inducible overexpression system for tagged PfSR1 and performed RNA immunoprecipitation followed by microarray analysis (RIP-chip) to recover and identify the endogenous RNA targets that bind PfSR1. Bioinformatic analysis of these RNAs revealed common sequence motifs potentially recognized by PfSR1. RNA-EMSAs show that PfSR1 preferentially binds RNA molecules containing these motifs. Interestingly, we find that PfSR1 not only regulates AS but also the steady-state levels of mRNAs containing these motifs in vivo.

Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state.

The human malaria parasite Plasmodium falciparum is auxotrophic for most amino acids. Its amino acid needs are met largely through the degradation of host erythrocyte hemoglobin; however the parasite must acquire isoleucine exogenously, because this amino acid is not present in adult human hemoglobin. We report that when isoleucine is withdrawn from the culture medium of intraerythrocytic P. falciparum, the parasite slows its metabolism and progresses through its developmental cycle at a reduced rate. Isoleucine-starved parasites remain viable for 72 h and resume rapid growth upon resupplementation. Protein degradation during starvation is important for maintenance of this hibernatory state. Microarray analysis of starved parasites revealed a 60% decrease in the rate of progression through the normal transcriptional program but no other apparent stress response. Plasmodium parasites do not possess a TOR nutrient-sensing pathway and have only a rudimentary amino acid starvation-sensing eukaryotic initiation factor 2α (eIF2α) stress response. Isoleucine deprivation results in GCN2-mediated phosphorylation of eIF2α, but kinase-knockout clones still are able to hibernate and recover, indicating that this pathway does not directly promote survival during isoleucine starvation. We conclude that P. falciparum, in the absence of canonical eukaryotic nutrient stress-response pathways, can cope with an inconsistent bloodstream amino acid supply by hibernating and waiting for more nutrient to be provided.

Epigenetic switches in clag3 genes mediate blasticidin S resistance in malaria parasites.

Malaria parasites induce changes in the permeability of the infected erythrocyte membrane to numerous solutes, including toxic compounds. In Plasmodium falciparum, this is mainly mediated by PSAC, a broad-selectivity channel that requires the product of parasite clag3 genes for its activity. The two paralogous clag3 genes, clag3.1 and clag3.2, can be silenced by epigenetic mechanisms and show mutually exclusive expression. Here we show that resistance to the antibiotic blasticidin S (BSD) is associated with switches in the expression of these genes that result in altered solute uptake. Low concentrations of the drug selected parasites that switched from clag3.2 to clag3.1 expression, implying that expression of one or the other clag3 gene confers different transport efficiency to PSAC for some solutes. Selection with higher BSD concentrations resulted in simultaneous silencing of both clag3 genes, which severely compromises PSAC formation as demonstrated by blocked uptake of other PSAC substrates. Changes in the expression of clag3 genes were not accompanied by large genetic rearrangements or mutations at the clag3 loci or elsewhere in the genome. These results demonstrate that malaria parasites can become resistant to toxic compounds such as drugs by epigenetic switches in the expression of genes necessary for the formation of solute channels.

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

A Plasmodium falciparum histone deacetylase regulates antigenic variation and gametocyte conversion.

The asexual forms of the malaria parasite Plasmodium falciparum are adapted for chronic persistence in human red blood cells, continuously evading host immunity using epigenetically regulated antigenic variation of virulence-associated genes. Parasite survival on a population level also requires differentiation into sexual forms, an obligatory step for further human transmission. We reveal that the essential nuclear gene, P. falciparum histone deacetylase 2 (PfHda2), is a global silencer of virulence gene expression and controls the frequency of switching from the asexual cycle to sexual development. PfHda2 depletion leads to dysregulated expression of both virulence-associated var genes and PfAP2-g, a transcription factor controlling sexual conversion, and is accompanied by increases in gametocytogenesis. Mathematical modeling further indicates that PfHda2 has likely evolved to optimize the parasite's infectious period by achieving low frequencies of virulence gene expression switching and sexual conversion. This common regulation of cellular transcriptional programs mechanistically links parasite transmissibility and virulence.

Whole-genome analysis of Plasmodium spp. Utilizing a new agilent technologies DNA microarray platform.

The application of DNA microarray technologies to malaria genomics has been widely used but has been limited by sample availability and technical variability. To address these issues, we present a microarray hybridization protocol that has been optimized for use with two new Agilent Technologies DNA microarrays for Plasmodium falciparum and P. berghei. Using the most recent genome sequences available for each species, we have designed ∼14,000 oligonucleotide probes representing ∼5,600 transcripts for each species. Included in each array design are numerous probes that allow for the identification of parasite developmental stages, common Plasmodium molecular markers used in genetic manipulation, and manufacturer probes that control for array consistency and quality. Overall, the Agilent Plasmodium spp. array designs and hybridization methodology provides a sensitive, easy-to-use, high-quality, cost-effective alternative to other currently available microarray platforms.