PTEX helps efficiently traffic haemoglobinases to the food vacuole inPlasmodium falciparum

Abstract

AbstractA key element ofPlasmodiumbiology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-containing protein complex termed thePlasmodiumtranslocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, bothin vitroandin vivo, suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathway, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which metabolic pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon’s involvement in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a another mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.Author summaryPlasmodium falciparumis the causative agent of the most severe form of malaria in humans, where the symptoms of the disease are derived from the continuous asexual replication of the parasite within the human red blood cells (RBCs) it infects. To survive within this niche, the parasite exports hundreds of parasite effector proteins across the vacuole it resides within and into the RBC. About a quarter of the exported proteins appear to be essential during the blood stage but the functions of these proteins largely remain uncharacterised. Protein export is facilitated by an essential protein complex termed thePlasmodiumtranslocon of exported proteins (PTEX). Conditional depletion of PTEX’s core components results in rapid parasite death presumably because essential proteins do not reach their functional destination in the RBC and their associated metabolic functions cannot be performed. To uncover what these essential metabolic functions are we knocked down PTEX150, a core component of PTEX. Metabolic analysis of the knockdown parasites indicated that haemoglobin (Hb) digestion was inhibited resulting in a reduction of Hb derived peptides, which serve as an amino acid source for the parasite. We determined that knocking down HSP101, another PTEX core component, also disrupted the Hb digestion pathway. Furthermore, we provide evidence that reduction of Hb digestion might be due to the failure to efficiently deliver early acting Hb digesting proteases to the cytostome, a specialised location where vesicles of Hb are taken into the parasite. PTEX may therefore play a role in delivering Hb proteases to the cytostome.