A crucial role for the C-terminal domain of exported protein 1 during the mosquito and hepatic stages of the Plasmodium berghei life cycle.

Intracellular Plasmodium parasites develop inside a parasitophorous vacuole (PV), a specialised compartment enclosed by a membrane (PVM) that contains proteins of both host and parasite origin. Although exported protein 1 (EXP1) is one of the earliest described parasitic PVM proteins, its function throughout the Plasmodium life cycle remains insufficiently understood. Here, we show that whereas the N-terminus of Plasmodium berghei EXP1 (PbEXP1) is essential for parasite survival in the blood, parasites lacking PbEXP1's entire C-terminal (CT) domain replicate normally in the blood but cause less severe pathology than their wild-type counterparts. Moreover, truncation of PbEXP1's CT domain not only impairs parasite development in the mosquito but also abrogates PbEXP1 localization to the PVM of intrahepatic parasites, severely limiting their replication and preventing their egress into the blood. Our findings highlight the importance of EXP1 during the Plasmodium life cycle and identify this protein as a promising target for antiplasmodial intervention.

Phosphomimetic substitution at Ser-33 of the chloroquine resistance transporter PfCRT reconstitutes drug responses in Plasmodium falciparum.

The chloroquine resistance transporter PfCRT of the human malaria parasite Plasmodium falciparum confers resistance to the former first-line antimalarial drug chloroquine, and it modulates the responsiveness to a wide range of quinoline and quinoline-like compounds. PfCRT is post-translationally modified by phosphorylation, palmitoylation, and, possibly, ubiquitination. However, the impact of these post-translational modifications on P. falciparum biology and, in particular, the drug resistance-conferring activity of PfCRT has remained elusive. Here, we confirm phosphorylation at Ser-33 and Ser-411 of PfCRT of the chloroquine-resistant P. falciparum strain Dd2 and show that kinase inhibitors can sensitize drug responsiveness. Using CRISPR/Cas9 genome editing to generate genetically engineered PfCRT variants in the parasite, we further show that substituting Ser-33 with alanine reduced chloroquine and quinine resistance by ∼50% compared with the parental P. falciparum strain Dd2, whereas the phosphomimetic amino acid aspartic acid could fully and glutamic acid could partially reconstitute the level of chloroquine/quinine resistance. Transport studies conducted in the parasite and in PfCRT-expressing Xenopus laevis oocytes linked phosphomimetic substitution at Ser-33 to increased transport velocity. Our data are consistent with phosphorylation of Ser-33 relieving an autoinhibitory intramolecular interaction within PfCRT, leading to a stimulated drug transport activity. Our findings shed additional light on the function of PfCRT and suggest that chloroquine could be reevaluated as an antimalarial drug by targeting the kinase in P. falciparum that phosphorylates Ser-33 of PfCRT.

The Plasmodium liver-stage parasitophorous vacuole: A front-line of communication between parasite and host.

The intracellular development and differentiation of the Plasmodium parasite in the host liver is a prerequisite for the actual onset of malaria disease pathology. Since liver-stage infection is clinically silent and can be completely eliminated by sterilizing immune responses, it is a promising target for urgently needed innovative antimalarial drugs and/or vaccines. Discovered more than 65 years ago, these stages remain poorly understood regarding their molecular repertoire and interaction with their host cells in comparison to the pathogenic erythrocytic stages. The differentiating and replicative intrahepatic parasite resides in a membranous compartment called the parasitophorous vacuole, separating it from the host-cell cytoplasm. Here we outline seminal work that contributed to our present understanding of the fundamental dynamic cellular processes of the intrahepatic malarial parasite with both specific host-cell factors and compartments.

Iron is a substrate of the Plasmodium falciparum chloroquine resistance transporter PfCRT in Xenopus oocytes.

The chloroquine resistance transporter of the human malaria parasite Plasmodium falciparum, PfCRT, is an important determinant of resistance to several quinoline and quinoline-like antimalarial drugs. PfCRT also plays an essential role in the physiology of the parasite during development inside erythrocytes. However, the function of this transporter besides its role in drug resistance is still unclear. Using electrophysiological and flux experiments conducted on PfCRT-expressing Xenopus laevis oocytes, we show here that both wild-type PfCRT and a PfCRT variant associated with chloroquine resistance transport both ferrous and ferric iron, albeit with different kinetics. In particular, we found that the ability to transport ferrous iron is reduced by the specific polymorphisms acquired by the PfCRT variant as a result of chloroquine selection. We further show that iron and chloroquine transport via PfCRT is electrogenic. If these findings in the Xenopus model extend to P. falciparum in vivo, our data suggest that PfCRT might play a role in iron homeostasis, which is essential for the parasite's development in erythrocytes.

Plasmodium berghei EXP-1 interacts with host Apolipoprotein H during Plasmodium liver-stage development.

The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wild-type parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies.

Oxidative insult can induce malaria-protective trait of sickle and fetal erythrocytes.

Plasmodium falciparum infections can cause severe malaria, but not every infected person develops life-threatening complications. In particular, carriers of the structural haemoglobinopathies S and C and infants are protected from severe disease. Protection is associated with impaired parasite-induced host actin reorganization, required for vesicular trafficking of parasite-encoded adhesins, and reduced cytoadherence of parasitized erythrocytes in the microvasculature. Here we show that aberrant host actin remodelling and the ensuing reduced cytoadherence result from a redox imbalance inherent to haemoglobinopathic and fetal erythrocytes. We further show that a transient oxidative insult to wild-type erythrocytes before infection with P. falciparum induces the phenotypic features associated with the protective trait of haemoglobinopathic and fetal erythrocytes. Moreover, pretreatment of mice with the pro-oxidative nutritional supplement menadione mitigate the development of experimental cerebral malaria. Our results identify redox imbalance as a causative principle of protection from severe malaria, which might inspire host-directed intervention strategies.

Ticket to ride: export of proteins to the Plasmodium falciparum-infected erythrocyte.

The malaria parasite Plasmodium falciparum exports numerous proteins to its chosen host cell, the mature human erythrocyte. Many of these proteins are important for parasite survival. To reach the host cell, parasites must cross multiple membrane barriers and then furthermore be targeted to their correct sub-cellular localisation. This novel transport pathway has received much research attention in the past decades, especially as many of the mechanisms are expected to be parasite-specific and thus potential targets for drug development. In this article we summarize some of the most recent advances in this field, and highlight areas in which further research is needed.