The chaperonin TRiC forms an oligomeric complex in the malaria parasite cytosol

Abstract

SUMMARY The malaria parasite exports numerous proteins into its host red blood cell (RBC). The trafficking of these exported effectors is complex. Proteins are first routed through the secretory system, into the parasitophorous vacuole (PV), a membranous compartment enclosing the parasite. Proteins are then translocated across the PV membrane in a process requiring ATP and unfolding. Once in the RBC compartment the exported proteins are then refolded and further trafficked to their final localizations. Chaperones are important in the unfolding and refolding processes. Recently, it was suggested that the parasite TRiC chaperonin complex is exported, and that it is involved in trafficking of exported effectors. Using a parasite-specific antibody and epitope-tagged transgenic parasites we could observe no export of Plasmodium TRiC into the RBC. We tested the importance of the parasite TRiC by creating a regulatable knockdown line of the TRiC-θ subunit. Loss of the parasite TRiC-θ led to a severe growth defect in asexual development, but did not alter protein export into the RBC. These observations indicate that the TRiC proteins play a critical role in parasite biology, though their function, within the parasite, appears unrelated to protein trafficking in the RBC compartment.

EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

“…Cpf1 from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) has been shown to mediate efficient genome editing in mammalian cells (Zetsche et al, ) and we tested both of these enzymes for their genome editing capacity in P. falciparum relative to a Cas9 editing system we previously developed (Garten et al, ; Spillman, Beck, Ganesan, Niles, & Goldberg, ). The 3′ region of exp1 was found to contain attractive Cas9 and Cpf1 target sites located in close proximity, providing an opportunity for initial Cpf1 testing (Figure 1a).…”

“…For Cas9‐mediated editing of the exp1 locus, the pAIO (Spillman et al, ) plasmid was first simplified by removing the yDHODH‐2A fusion to Cas9‐NLS‐FLAG using a QuikChange Lightning Multi Site Directed Mutagenesis kit (Agilent) and the primer P5, resulting in the plasmid pAIO2. The BtgZI site in the pre‐single guide RNA cassette was then replaced with AflII by inserting the annealed sense and antisense oligo pair P6/P7 into BtgZI, resulting in the plasmid pAIO3.…”

“…Editing of the 3 0 end of both loci was also successful ( Figure S2C,D); however, both ETRAMP10.2 apt and ETRAMP5 apt parasite lines were found to a have truncated 3 0 aptamer arrays (reduced from 10X to 6X, data not shown). Partial truncation of 3 0 aptamers rapidly diminishes translational control (Spillman et al, 2017) and anhydrotetracycline (aTc) washout did not result in measurable knockdown ( Figure S2C,D), thus ETRAMP10.2 and ETRAMP5 were not pursued further in this study.…”

Plasmodium falciparum Niemann-Pick Type C1-Related Protein is a Druggable Target Required for Parasite Membrane Homeostasis

“…An attempt to disrupt the pfncr1 gene using a CRISPR/Cas9-targeting approach did not succeed, suggesting an essential function during blood-stage malaria growth. Next, we created parasites in which pfncr1 expression is regulated by anhydrotetracyline (aTc) using the previously described TetR-DOZI/aptamer translational repression technology 15,16 (Supplementary Figs. S2A, S2B).…”

“…An AscI site was introduced at the 5' end and an AflII site was introduced at the 3' end. After generation of single homologous region fragments, RHR and LHR PCR products were mixed, amplified with primers RHR1F and LHR1R and cloned into the plasmid pMG75 as described 16 . The resulting construct (pMG75-PfNCR1) contains a single in frame HA sequence followed by 10x aptamers for aTc-regulatable translational repression.…”