APEX-based proximity labeling inPlasmodiumidentifies a membrane protein with dual functions during mosquito infection

Abstract

Transmission of the malaria parasite Plasmodium to mosquitoes necessitates gamete egress from red blood cells to allow zygote formation and ookinete motility to enable penetration of the midgut epithelium. Both processes are dependent on the secretion of proteins from distinct sets of specialized vesicles. Inhibiting some of these proteins has shown potential for blocking parasite transmission to the mosquito. To identify new transmission blocking vaccine candidates, we defined the microneme content from ookinetes of the rodent model organism Plasmodium berghei using APEX2-mediated rapid proximity-dependent biotinylation. Besides known proteins of ookinete micronemes, this identified over 50 novel candidates and sharpened the list of a previous survey based on subcellular fractionation. Functional analysis of a first candidate uncovered a dual role for this membrane protein in male gametogenesis and ookinete midgut traversal. Mutation of a putative trafficking motif in the C-terminus led to its mis-localization in ookinetes and affected ookinete to oocyst transition but not gamete formation. This suggests the existence of distinct functional and transport requirements for Plasmodium proteins in different parasite stages.SignificanceThe genome of the malaria parasite Plasmodium contains over 5500 genes, of which over 30% have no assigned function. Transmission of Plasmodium spp. to the mosquito contains several essential steps that can be inhibited by antibodies or chemical compounds. Yet few proteins involved in these processes are characterized, thus limiting our capacity to generate transmission interfering tools. Here, we establish a method to rapidly identify proteins in a specific compartment within the parasite that is essential for establishment of an infection within the mosquito, and identify over 50 novel candidate proteins. Functional analysis of the top candidate identifies a protein with two independent essential functions in subsequent steps along the Plasmodium life cycle within the mosquito.Highlightsfirst use of APEX based proximity ligation in Apicomplexaidentification of >50 putative ookinete surface proteinsnovel membrane protein essential for microgamete egress and ookinete migrationputative trafficking motif essential in ookinetes but not gametes

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

“…BioID approaches have been previously successfully applied to identify IMC proteins of T . gondii (Chen et al, 2015; Chen et al, 2017) as well as novel components of several Plasmodium organelles (Birnbaum et al, 2020; Boucher et al, 2018; Geiger et al, 2020; Kehrer et al, 2020; Kehrer, Frischknecht, & Mair, 2016; Khosh‐Naucke et al, 2018; Schnider, Bausch‐Fluck, Brühlmann, Heussler, & Burda, 2018).…”

A microtubule associated protein is essential for malaria parasite transmission

“…2017 (42). The amplicon was cloned into the pL18 vector (77) using EcoRI/BamHI restriction sites. Prior to transfection, the vector was linearized using the SwaI restriction enzyme followed by ethanol-precipitation.…”

Proximity‐dependent biotinylation approaches to study apicomplexan biology

“…Labeling with APEX2 has been used to determine the protein content of secretory vesicles in P. berghei ookinetes. To this end, the well-known micronemal protein SOAP was fused to APEX2, which led to the identification of known and novel putative micronemal proteins via MS analyses (Kehrer et al, 2020). In contrast to SOAP-APEX2, the fusion of SOAP to BirA* was not functional, possibly because of the long incubation time needed.…”

“…In contrast, the food vacuole of the parasite is strongly acidic (Homewood & Neame, 1974) and most likely an unsuitable environment for biotinylation. Compartments of the secretory pathway, such as ER, Golgi, and secretory vesicles, are only mildly acidic and their milieu does not seem to impair biotinylation (Wu et al, 2001;Kehrer et al, 2016Kehrer et al, , 2020Geiger et al, 2020;Kumar et al, 2021). Yet, in mammalian cells, biotinylation of the ER lumen was more efficient using TurboID compared with classic BirA* (May et al, 2020).…”

“…In short-lived parasite stages, such as Plasmodium ookinetes, BirA*-based PDB is problematic since the labeling takes more time than the development of the motile cell. Thus, biotinylation within ookinetes seems more efficient using the faster-acting peroxidase APEX2 (Kehrer et al, 2020). Optimized BirA* versions, such as TurboID or miniTurbo, with labeling times around 10 min (see Section 1.3; Branon et al, 2018) have not yet been used in Plasmodium parasites and might be alternatives to APEX2.…”