Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells

Abstract

Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside‐out mode during which the membrane of the parasitophorous vacuole (PV) ruptures prior to the erythrocyte membrane. Membrane rupture requires exocytosis of specialized egress vesicles of the parasites; that is, osmiophilic bodies (OBs) involved in rupturing the PV membrane, and vesicles that harbor the perforin‐like protein PPLP2 (here termed P‐EVs) required for erythrocyte lysis. While some OB proteins have been identified, like G377 and MDV1/Peg3, the majority of egress vesicle‐resident proteins is yet unknown. Here, we used high‐resolution imaging and BioID methods to study the two egress vesicle types in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the P‐EVs and that exocytosis of the P‐EVs, but not of the OBs, is calcium sensitive. Both vesicle types exhibit distinct proteomes with the majority of proteins located in the OBs. In addition to known egress‐related proteins, we identified novel components of OBs and P‐EVs, including vesicle‐trafficking proteins. Our data provide insight into the immense molecular machinery required for the inside‐out egress of P. falciparum gametocytes.

The Plasmodium falciparumCCCH zinc finger protein MD3 regulates male gametocytogenesis through its interaction with RNA‐binding proteins

“…To unveil the MD3 interactome in asexual and sexual blood stage parasites, we employed BioID-MS analyses, using vectors pARL-MD3-pfama1-GFP-BirA or pARL-MD3-pffnpa-GFP-BirA. While these vectors were previously successfully used for BioID analyses (Musabyimana et al, 2022;Sassmannshausen et al, 2023), it should be noted that pffnpa, the promotor of which was used to drive episomal MD3-GFP-BirA overexpression in gametocytes, is highly expressed in females (e.g., Lasonder et al, 2016;Scholz et al, 2008). The use of heterologous promoters in overexpression plasmids may in general affect the outcome of interactome analyses, in particular, when studying differential protein levels during male versus female gametocytogenesis.…”

“…To determine the MD3 interaction network, we generated transgenic lines episomally expressing an MD3-GFP-BirA fusion protein as described (Musabyimana et al, 2022;Sassmannshausen et al, 2023). Blood stage parasites were transfected with the vec-…”

Mechanisms of host cell exit by intracellular pathogens

“…Membrane destruction during host cell exit is particularly known for apicomplexan parasites. In this issue, various articles provide novel insight into molecules involved during host cell exit by Plasmodium and Toxoplasma, like phospholipases (Pietsch et al, 2023), zinc-finger proteins (Farrukh et al, 2023), cytoskeletal elements (Beyeler et al, 2023) or vesicle-resident factors (Sassmannshausen et al, 2023) and plasma membrane-bound proteins (Jennison et al, 2023;Thieleke-Matos et al, 2024). In addition, two reviews summarize apicomplexan exit strategies (Jimenéz-Ruiz et al, 2023;Scheiner et al, 2023).…”

Global Release of Translational Repression AcrossPlasmodium’sHost-to-Vector Transmission Event

“…We selected DOZI and ALBA4 as they are known to associate with the 5' or 3' end of bound mRNAs, respectively (10,20,56), and thus provide protein proxies for the two ends of target mRNAs. We initially tested the firstgeneration BioID enzyme before adopting the use of the further engineered TurboID (TID) ligase that allowed for more robust labeling in a shorter labeling time, as we and others have recently shown for P. falciparum and P. berghei (Fig S4BC) (47,(57)(58)(59)(60)(61). To establish an appropriate duration of labeling, we assessed in vivo biotinylation with supplementation with 150 µM biotin for increasing time, then used total parasite lysate for affinity blots probed with streptavidin-HRP for both bait parasite lines, ALBA4::TurboID::GFP and DOZI::TurboID::GFP (Fig S4B -F).…”