A Conserved Region in the EBL Proteins Is Implicated in Microneme Targeting of the Malaria ParasitePlasmodium falciparum

Abstract

The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBA-GFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.Plasmodium falciparum is the causative agent of the most severe form of human malaria. Increasing global prevalence of malaria is reported, and it is estimated that mortality reaches 1-2 million per year, with the biggest impact on young African children (1). The invasion, host cell modification and subsequent destruction of erythrocytes by the obligatory intracellular parasite are responsible for the pathobiology in the human host. The invasion process is initiated by merozoites, polarorganized, ϳ1.8 m, slightly elongated invasive cells (2, 3). Secretory organelles including the micronemes and rhoptries (4) store and secrete proteins that enable the parasite to (i) adhere to surface receptors of the host cell, (ii) invade the new host cell, and (iii) establish itself within the parasitophorous vacuole (5). Because of compartmentalization, this small protozoan parasite relies on a sophisticated secretory system that delivers proteins to multiple subcellular destinations (6 -11).Trafficking is initiated by classical N-terminal signal sequences that direct proteins across the endoplasmic reticulum (ER) into the secretory pathway (12). In the Golgi and trans-Golgi network (TGN) the proteins are routed either to constitutive or regulated secretory pathways (13). Once the proteins reach the Golgi exit face, they are embedded into transport vesicles according to sequence information or by an acquired label and brought to their final destinations (14). Proteins entering the regulated pathways are sorted to secretory compartments and are released upon a stimulus (15). One intriguing question concerns the nature of the signal(s) necessary and sufficient for post-Golgi protein sorting to the predetermined destinations. In mammalian cells the transport of transmembrane proteins to endosomes and lysosomes is most commonly mediated by distinct cytoplasmic-sorting motifs (16,17). This has also been shown in the protozoan Toxoplasma gondii, a Plasmodium-related parasite. T...

The Role of Palmitoylation for Protein Recruitment to the Inner Membrane Complex of the Malaria Parasite

“…PCR amplicons were digested with KpnI and AvrII, cloned into a derivative of pARL-1a-GFP. Expression of the GFP fusion construct is controlled by the late stage promoter of ama1 (32). To circumvent the internal KpnI site in the genes coding PfDHHC3 and PfDHHC9, both open reading frames were cloned into the BamHI/SalI of a derivative of pBcamR (33).…”

“…2C). The stage-specific expression pattern of PfDHHC1 was analyzed using synchronized 3D7-PfDHHC1-GFP parasite material harvested 8,16,24,32,40, and 48 h postinvasion and probed with either anti-GFP or anti-GAPM2 antibodies as a stage-specific control in a Western blot (Fig. 2D).…”

Far from the Madding Crowd: the Molecular Basis for Immunological Escape ofPlasmodium falciparum

“…Regarding EBL cHABPs, there is cadence where ~80 ± 20 residues are separated, as occurs in EBA-175 and EBA-140 (and EBA-181, though lacking 3D structure), suggesting that EBL p r o t e i n s h a v e v e r y s i m i l a r s t r u c t u r a l characteristics due to the presence of cysteinerich Duffy binding-like (DBL) domains which are responsible for creating multiple binding sites for recognition and binding to erythrocyte receptor (Tolia et al, 2005;Treeck et al, 2006). This occurs with EBA-175 cHABP 1783 ( 580 HRNK KNDKLYRDEWWKVIKK 599 ) that binds to glycophorin A receptor on RBC and EBA-140 cHABP 26144 ( 541 DLADIIKGSDIIKDYYGKKM 560 ) that interacts with RBC glycophorin C.…”

Mononeme: A new secretory organelle in Plasmodium falciparum merozoites identified by localization of rhomboid-1 protease

“…These organelles were first identified by their distinct morphological appearances in transmission electron microscopy (2-4). Many parasite proteins required for invasion of erythrocytes are segregated into the micronemes (5,6). For example, Plasmodium falciparum apical membrane antigen 1 (AMA1) is held in the micronemes in merozoites inside of erythrocytes.…”