Plasmodium falciparumOTU-like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with noncanonical role of Atg8

Abstract

The metabolic pathways associated with the mitochondrion and the apicoplast in Plasmodium, 2 parasite organelles of prokaryotic origin, are considered as suitable drug targets. In the present study, we have identified functional role of a novel ovarian tumour unit (OTU) domain-containing cysteine protease of Plasmodium falciparum (PfOTU). A C-terminal regulatable fluorescent affinity tag on native protein was utilised for its localization and functional characterization. Detailed studies showed vesicular localization of PfOTU and its association with the apicoplast. Degradation-tag mediated knockdown of PfOTU resulted in abnormal apicoplast development and blocked development of parasites beyond early-schizont stages in subsequent cell cycle; downregulation of PfOTU hindered apicoplast protein import. Further, the isoprenoid precursor-mediated parasite growth-rescue experiments confirmed that PfOTU knockdown specifically effect development of functional apicoplast. We also provide evidence for a possible biological function of PfOTU in membrane deconjugation of Atg8, which may be linked with the apicoplast protein import. Overall, our results show that the PfOTU is involved in apicoplast homeostasis and associates with the noncanonical function of Atg8 in maintenance of parasite apicoplast.

Autophagy-Related Protein ATG18 Regulates Apicoplast Biogenesis in Apicomplexan Parasites

“…We found that PfATG8 resides in vesicular structures and while most of these vesicles were in close proximity of the branching organelle, some vesicles were coincident ( Fig. 6A ) as observed previously ( 13 , 22 ). The depletion of PfATG18 resulted in a significant loss of punctate localization of PfATG8 along with an increase in diffused cytoplasmic localization ( Fig.…”

“…2B ) and in proximity of vesicles containing downstream autophagy protein PfATG8 ( Fig. 2C ), which was transiently localized to the apicoplast in previous studies ( 13 , 22 ) (see Fig. 6A ).…”

“…While genetic disruption of PfATG8 has not been reported yet, it has been speculated in apicoplast-related function on the basis of localization studies. While in some studies reasonable colocalization of PfATG8 with the apicoplast was observed, in other cases only some of the PfATG8 puncta overlapped with the apicoplast ( 13 , 22 , 29 ). We found PfATG8 mainly in vesicles around the apicoplast ( Fig.…”

“…Plasmodium falciparum antibodies were anti- P . falciparum Crt (anti-PfCrt) (MR4, rabbit, 1:400), anti-ATG8 for IFA (mouse, 1:200) ( 22 ), anti-ClpP (rabbit, 1:300), anti-glutathione S -transferase (anti-GST) (mouse, 1:5,000), anti-ACP (diluted 1:1,000 for IFA) (antibody was a kind gift from Geoff McFadden), and anti- P . falciparum ATG8 (anti-PfATG8) for Western blots (rabbit, 1:500) (a gift from Puran Sijwali).…”

Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part III: Protozoa (2): Plasmodium spp.

“…Gene knockout showed abnormal apicoplast development which resulted in subsequent arrest of early-schizont developmental stages in further life cycles. It was observed that PfOTU is an essential CP in apicoplast protein import, and is significantly linked with apicoplast homeostasis [89] .…”