New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach.

Malaria, caused by Plasmodium falciparum (P. falciparum), ranks as one of the most baleful infectious diseases worldwide. New antimalarial treatments are needed to face existing or emerging drug resistant strains. Protein degradation appears to play a significant role during the asexual intraerythrocytic developmental cycle (IDC) of P. falciparum. Inhibition of the ubiquitin proteasome system (UPS), a major intracellular proteolytic pathway, effectively reduces infection and parasite replication. P. falciparum and erythrocyte UPS coexist during IDC but the nature of their relationship is largely unknown. We used an approach based on Tandem Ubiquitin-Binding Entities (TUBEs) and 1D gel electrophoresis followed by mass spectrometry to identify major components of the TUBEs-associated ubiquitin proteome of both host and parasite during ring, trophozoite and schizont stages. Ring-exported protein (REX1), a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, was found to reach a maximum level of ubiquitylation in trophozoites stage. The Homo sapiens (H. sapiens) TUBEs associated ubiquitin proteome decreased during the infection, whereas the equivalent P. falciparum TUBEs-associated ubiquitin proteome counterpart increased. Major cellular processes such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. BIOLOGICAL SIGNIFICANCE: In this work we analyze for the first time the interconnection between Plasmodium and human red blood cells ubiquitin-regulated proteins in the context of infection. We identified a number of human and Plasmodium proteins whose ubiquitylation pattern changes during the asexual infective stage. We demonstrate that ubiquitylation of REX1, a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, peaks in trophozoites stage. The ubiquitin-proteome from P. falciparum infected red blood cells (iRBCs) revealed a significant host-parasite crosstalk, underlining the importance of ubiquitin-regulated proteolytic activities during the intraerythrocytic developmental cycle (IDC) of P. falciparum. Major cellular processes defined from gene ontology such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. Given the importance of ubiquitylation in the development of infectious diseases, this work provides a number of potential drug-target candidates that should be further explored.

Case Study of Small Molecules As Antimalarials: 2-Amino-1-phenylethanol (APE) Derivatives.

Antiparasitic oral drugs have been associated to lipophilic molecules due to their intrinsic permeability. However, these kind of molecules are associated to numerous adverse effects, which have been extensively studied. Within the Tres Cantos Antimalarial Set (TCAMS) we have identified two small, soluble and simple hits that even presenting antiplasmodial activities in the range of 0.4-0.5 µM are able to show in vivo activity.

Magnetic isolation of Plasmodium falciparum schizonts iRBCs to generate a high parasitaemia and synchronized in vitro culture.

BACKGROUND: The establishment of methods for an in vitro continuous culture of Plasmodium falciparum is essential for gaining knowledge into its biology and for the development of new treatments. Previously, several techniques have been used to synchronize, enrich and concentrate P. falciparum, although obtaining cultures with high parasitaemia continues being a challenging process. Current methods produce high parasitaemia levels of synchronized P. falciparum cultures by frequent changes of culture medium or reducing the haematocrit. However, these methods are time consuming and sometimes lead to the loss of synchrony. METHODS: A procedure that combines Percoll and sorbitol treatments, the use of magnetic columns, and the optimization of the in vitro culture conditions to reach high parasitaemia levels for synchronized Plasmodium falciparum cultures is described. RESULTS: A new procedure has been established using P. falciparum 3D7, combining previous reported methodologies to achieve in vitro parasite cultures that reach parasitaemia up to 40% at any intra-erythrocytic stage. High parasitaemia levels are obtained only one day after magnetic column purification without compromising the parasite viability and synchrony. CONCLUSIONS: The described procedure allows obtaining a large scale synchronized parasite culture at a high parasitaemia with less manipulations than other methods previously described.

Identification and validation of tetracyclic benzothiazepines as Plasmodium falciparum cytochrome bc1 inhibitors.

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc(1) complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc(1) activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.