Functions have yet to be defined for the majority of genes of Plasmodium falciparum, the agent responsible for the most serious form of human malaria. Here we report changes in P. falciparum gene expression induced by 20 compounds that inhibit growth of the schizont stage of the intraerythrocytic development cycle. In contrast with previous studies, which reported only minimal changes in response to chemically induced perturbations of P. falciparum growth, we find that approximately 59% of its coding genes display over three-fold changes in expression in response to at least one of the chemicals we tested. We use this compendium for guilt-by-association prediction of protein function using an interaction network constructed from gene co-expression, sequence homology, domain-domain and yeast two-hybrid data. The subcellular localizations of 31 of 42 proteins linked with merozoite invasion is consistent with their role in this process, a key target for malaria control. Our network may facilitate identification of novel antimalarial drugs and vaccines.

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

SummaryPlasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX. Previously PTEX150, HSP101 and EXP2 have been shown to be bona fide members of PTEX. Here we validate that PTEX88 and TRX2 are also genuine members of PTEX and provide evidence that expression of PTEX components are also expressed in early gametocytes, mosquito and liver stages, consistent with observations that protein export is not restricted to asexual stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in Plasmodium berghei, in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth. In contrast, the putative thioredoxin-like protein TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro, and reduced capacity to cause severe disease in a cerebral malaria model. Thus, while not essential for parasite survival, TRX2 may help to optimize PTEX activity. Importantly, the generation of TRX2 knockout parasites that display altered phenotypes provides a much-needed tool to dissect PTEX function.

SummaryA short motif termed Plasmodium export element (PEXEL) or vacuolar targeting signal (VTS) characterizes Plasmodium proteins exported into the host cell. These proteins mediate host cell modifications essential for parasite survival and virulence. However, several PEXEL-negative exported proteins indicate that the currently predicted malaria exportome is not complete and it is unknown whether and how these proteins relate to PEXEL-positive export. Here we show that the N-terminal 10 amino acids of the PEXEL-negative exported protein REX2 (ringexported protein 2) are necessary for its targeting and that a single-point mutation in this region abolishes export. Furthermore we show that the REX2 transmembrane domain is also essential for export and that together with the N-terminal region it is sufficient to promote export of another protein. An N-terminal region and the transmembrane domain of the unrelated PEXEL-negative exported protein SBP1 (skeleton-binding protein 1) can functionally replace the corresponding regions in REX2, suggesting that these sequence features are also present in other PEXEL-negative exported proteins. Similar to PEXEL proteins we find that REX2 is processed, but in contrast, detect no evidence for N-terminal acetylation.

Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity, and ultimately pathogenesis of Plasmodium falciparum rely on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here, we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor’s mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future anti-malarials targeting both the glideosome motor and its regulatory elements.