PfClpC Is An Essential Clp Chaperone Required For Plastid Integrity And Clp Protease Stability In Plasmodium falciparum

Abstract

SummaryThe deadly malaria parasite, Plasmodium falciparum, contains a non-photosynthetic plastid known as the apicoplast, that functions to produce essential metabolites. Little is known about its biology or regulation, but drugs that target the apicoplast are clinically effective. Using phylogenetic analysis, we identified a putative complex of clp (caseinolytic protease) genes. We genetically targeted members of this complex and generated conditional mutants of the PfClpC chaperone and PfClpP protease and found that they co-localize in the apicoplast. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss, and was rescued by addition of the essential apicoplast-derived metabolite, IPP. Using a double conditional-mutant parasite line, we discovered that the chaperone activity is required to stabilize the active protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex.. CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

“…To isolate rare P. falciparum mutants that have lost their apicoplast due to biogenesis defects, we set out to design a live-cell reporter for apicoplast loss. The apicoplast contains a prokaryotic caseinolytic protease (Clp) system composed of a chaperone ClpC that recognizes and unfolds substrates and a protease ClpP that degrades the recognized substrates (El Bakkouri et al, 2010;Florentin et al, 2017;Rathore et al, 2010). We hypothesized that (1) in the presence of a functional apicoplast, ClpCP could be co-opted to degrade and turn 'off' a fluorescent reporter, whereas (2) loss of the apicoplast would result in loss of ClpCP activity and therefore turn 'on' the reporter ( Figure 1A).…”

An ER CREC family protein regulates the egress proteolytic cascade in malaria parasites

“…Plasmodium parasites were cultured in RPMI 1640 medium supplemented with Albumax I (Gibco) and transfected as described earlier (69–72). T. gondii tachyzoites were maintained in vitro by serial passage in Dulbecco’s modified minimal essential media (DMEM) with 1% FBS, 2.5 μg/ml amphotericin B, and 100 μg/ml streptomycin.…”

“…Constructs utilized in this study were confirmed by sequencing. PCR products were inserted into the respective plasmids using the In-Fusion cloning system (Clontech), the sequence- and ligation-independent cloning (SLIC) method (71, 72), T4-ligation (New England BioLabs), or site-directed mutagenesis using QuickChange (Agilent). To generate the pHA-SDEL- glmS/M9 plasmid, primers 7+8 were used to add an SDEL sequence at the end of the HA tag in pHA- glmS and pHA- M9 plasmids (71, 72).…”

“…Asynchronous growth assays were done as described previously (71, 72). Briefly, 5mM glucosamine (GlcN) (Sigma) was added to the growth medium and parasitemia was monitored every 24hrs via flow cytometry using a CyAn ADP (Beckman Coulter) or CytoFLEX (Beckman Coulter) instrument and analyzed by FlowJo software (Treestar, Inc.).…”

“…Western blotting for Plasmodium parasites was performed as described previously (71, 72). Briefly, parasites were permeabilized selectively by treatment with ice-cold 0.04% saponin in PBS for 10 min and pellets were collected for detection of proteins with the parasite.…”