Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.

About half the world's population is at risk of malaria, with Plasmodium falciparum malaria being responsible for the most malaria related deaths globally. Antimalarial drugs such as chloroquine and artemisinin are directed towards the proliferating intra-erythrocytic stages of the parasite, which is responsible for all the clinical symptoms of the disease. These antimalarial drugs have been reported to function via multiple pathways, one of which induces DNA damage via the generation of free radicals and reactive oxygen species. An urgent need to understand the mechanistic details of drug response and resistance is highlighted by the decreasing clinical efficacy of the front line drug, Artemisinin. The replication factor C subunit 1 is an important component of the DNA replication machinery and DNA damage response mechanism. Here we show the translocation of PfRFC1 from an intranuclear localisation to the nuclear periphery, indicating an orchestrated progression of distinct patterns of replication in the developing parasites. PfRFC1 responds to genotoxic stress via elevated protein levels in soluble and chromatin bound fractions. Reduction of PfRFC1 protein levels upon treatment with antimalarials suggests an interplay of replication, apoptosis and DNA repair pathways leading to cell death. Additionally, mislocalisation of the endogenously tagged protein confirmed its essential role in parasites' replication and DNA repair. This study provides key insights into DNA replication, DNA damage response and cell death in P. falciparum.

The microtubule cytoskeleton forms the most prominent structural system in Trypanosoma brucei , undergoing extensive modifications during the cell cycle. Visualization of tyrosinated microtubules leads to a semiconservative mode of inheritance, whereas recent studies employing microtubule plus end tracking proteins have hinted at an asymmetric pattern of cytoskeletal inheritance. To further the knowledge of microtubule synthesis and inheritance during T. brucei cell cycle, the dynamics of the microtubule cytoskeleton was visualized by inducible YFP- α -tubulin expression. During new flagellum/flagellum attachment zone (FAZ) biogenesis and cell growth, YFP- α -tubulin was incorporated mainly between the old and new flagellum/FAZ complexes. Cytoskeletal modifications at the posterior end of the cells were observed with EB1, a microtubule plus end binding protein, particularly during mitosis. Additionally, the newly formed microtubules segregated asymmetrically, with the daughter cell inheriting the new flagellum/FAZ complex retaining most of the new microtubules. Together, our results suggest an intimate connection between new microtubule formation and new FAZ assembly, consequently leading to asymmetric microtubule inheritance and cell division.

Trypanosoma brucei is a parasite that causes African sleeping sickness in humans and nagana in livestock and is transmitted by the tsetse fly. There is an urgent need for the development of new drugs against African trypanosomiasis due to the lack of vaccines and effective drugs. Orlistat (also called tetrahydrolipstatin or THL) is an FDA-approved antiobesity drug targeting primarily the pancreatic and gastric lipases within the gastrointestinal tract. It shows potential activities against tumors, mycobacteria, and parasites. Herein, we report the synthesis and evaluation of an expanded set of orlistat-like compounds, some of which showed highly potent trypanocidal activities in both the bloodstream form (BSF) and the procyclic form (PCF) of T. brucei. Subsequent in situ parasite-based proteome profiling was carried out to elucidate potential cellular targets of the drug in both forms. Some newly identified targets were further validated by the labeling of recombinantly expressed enzymes in Escherichia coli lysates. Bioimaging experiments with a selected compound were carried out to study the cellular uptake of the drug in T. brucei. Results indicated that orlistat is much more efficiently taken up by the BSF than the PCF of T. brucei and has clear effects on the morphology of mitochondria, glycosomes, and the endoplasmic reticulum in both BSF and PCF cells. These results support specific effects of orlistat on these organelles and correlate well with our in situ proteome profiling. Given the economic challenges of de novo drug development for neglected diseases, we hope that our findings will stimulate further research towards the conversion of orlistat-like compounds into new trypanocidal drugs.