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

Abstract

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of twelve known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represent structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites.

Gene-by-gene screen of the unknown proteins encoded onP. falciparumchromosome 3

“…Co-expression with apical markers revealed no co-localisation with micronemes, rhoptry bulb or neck but a location even more apical than all these markers (Figure S1G), indicating that Chr3C13 is a novel apical protein located close to the apical tip. Chr3C12 was recently located at the inner membrane complex (IMC) (named PIC4) using this cell line (Wichers et al, 2021). Chr3C19 and Chr3C20 showed a localisation pattern reminiscent of the mitochondrion, however, only Chr3C19 (Figure 1A) co-localised with Mito-Tracker (Figure S1H, I).…”

A conserved Plasmodium structural integrity maintenance protein (SIMP) is associated with sporozoite membrane and is essential for maintaining shape and infectivity

“…The mutant sporozoites were stained with FITC conjugated phalloidin (that binds to filamentous actin) and 3D11. To study the organization of the inner membrane complex (IMC), an IMC marker (Wichers et al, 2021) anti‐PIC5 (rabbit polyclonal) antibody was used in combination with 3D11. To confirm the possible orientation of the Pb SIMP C‐terminus, IFA was performed under permeabilized and non‐permeabilized conditions.…”

“…Since sporozoite motility is dependent on the traction generated by a molecular motor that is connected with the inner membrane complex (IMC), we also analyzed the organization of IMC in mutant sporozoites. Immunostaining of PIC5, an IMC marker(Wichers et al, 2021) revealed an intact IMC organization in mutants (Figure5b).…”

Characterization of apicomplexan amino acid transporters (ApiATs) in the malaria parasite Plasmodium falciparum

“…For endogenous tagging using the SLI system 38 and glmS based conditional knockdown 40 a 855 ( Pf ApiAT4/MFR5/PF3D7_1129900) and 1001 bp ( Pf ApiAT2/MFR4/PF3D7_0914700) homology region was amplified using 3D7 gDNA and cloned into pSLI-PIC1-GFP-glmS 61 using the NotI/MluI restriction site.…”

“…For overexpression constructs the full length Pf ApiAT10 sequence was amplified from parasite gDNA and cloned into pARL-ama1 46 -AIP-mCherry-yDHODH 61 using the XhoI/KpnI restriction site or into the pARL-crt 45 -PF3D7_0324600-GFP-hDHFR 37 plasmid using the KpnI/AvrII restriction site. Oligonucleotides used to generate the DNA fragments are summarized in Table S1.…”