Samuel Pazicky

The zoonotic parasite Plasmodium knowlesi has emerged as a widespread cause of human malaria in Southeast Asia. We assembled a high-resolution transcriptome of zoonotic P. knowlesi across its intraerythrocytic developmental cycle and combined transcriptomics, quantitative proteomics and thermal stability measurements to compare parasites cultured in macaque and human red blood cells. We found that P. knowlesi accelerates through the ring stage to settle at the same developmental pace as P. falciparum during the trophozoite and schizont stages. Subsequent multi-omic analysis revealed broad transcriptional and proteomic adaptations of P. knowlesi to the human host cell environment. Parasites cultured in human cells expressed glutathione synthetase earlier and displayed changes in upstream processes that include NADPH production by the pentose phosphate pathway and glutamine synthesis. These changes were accompanied by further differences in the expression of ribosomal subunits and mitochondrial respiratory complexes. Together, our findings offer a systems-level view of non-genetic adaptations of P. knowlesi to the human red blood cell environment.

The zoonotic parasite Plasmodium knowlesi has emerged as a widespread cause of human malaria in Southeast Asia. We assembled a high-resolution transcriptome of zoonotic P. knowlesi across its intraerythrocytic developmental cycle and combined transcriptomics, quantitative proteomics and thermal stability measurements to compare parasites cultured in macaque and human red blood cells. We found that P. knowlesi accelerates through the ring stage to settle at the same developmental pace as P. falciparum during the trophozoite and schizont stages. Subsequent multi-omic analysis revealed broad transcriptional and proteomic adaptations of P. knowlesi to the human host cell environment. Parasites cultured in human cells expressed glutathione synthetase earlier and displayed changes in upstream processes that include NADPH production by the pentose phosphate pathway and glutamine synthesis. These changes were accompanied by further differences in the expression of ribosomal subunits and mitochondrial respiratory complexes. Together, our findings offer a systems-level view of non-genetic adaptations of P. knowlesi to the human red blood cell environment.

Spreading resistance to clinically used antimalarial drugs increases the need for identification of new drug targets. Here, we screened 25 antimalarials with known and unknown mode of action to validate old and find new drug targets in P. falciparum . Combining experimental approach by proteome-wide cellular thermal shift assay and computational filtering by molecular docking, we found the drugs to bind to previously known drug targets, validated ACS10 as the target of MMV665915 and discovered new drug targets. Furthermore, we updated the experimental and analytical MS-CETSA pipeline with the inclusion of membrane solubilization step, validated the targets of atovaquone and cipargamin on cell lysates and in intact cells and mapped the parasite response to these antimalarials, identifying monocarboxylate transporter MCP2 as atovaquone transporter.

The malaria parasite Plasmodium falciparum undergoes a complex intraerythrocytic developmental cycle (IDC) that relies on a dynamic network of protein-protein interactions. These are usually mapped ex vivo, limiting our understanding of their dynamics and composition in natural environments. Here we introduce the meltome-assisted profiling of protein complexes (MAP-X) that maps the complexome through thermal proteome profiling in intact cells. We applied MAP-X across seven timepoints in the P. falciparum IDC. MAP-X predicted more than 20,000 interactions, resolving conserved protein complexes, reproducing previously identified interactions and finding previously unreported associations. We found that malaria protein complexes undergo distinct dynamic alterations, and we predicted their moonlighting subunits that dissociate from their native complex to assume different biological functions. Altogether, our findings provide a resource for uncovering Plasmodium biology and show that MAP-X can characterize protein complexes in intact cells to reveal cellular physiology at a proteome-wide level.

As the decline of malaria cases stalled over the last five years, novel targets in Plasmodium falciparum are necessary for the development of new drugs. Glycogen Synthase Kinase (PfGSK3) has been identified as a potential target, since its selective inhibitors were shown to disrupt the parasite’s life cycle. Here, we show that PfGSK3 exhibits autophosphorylation, leading to an extensive phosphorylation both in vitro and in the parasite. In the uncanonical N-terminal region of the parasite enzyme, we identified several autophosphorylation sites that regulate the activity of PfGSK3. By combining molecular modeling with experimental small-angle X-ray scattering data, we show that increased PfGSK3 activity is promoted by conformational changes in the PfGSK3 N-terminus, triggered by N-terminal phosphorylation. Our work provides novel insights into the structure and regulation of the malarial PfGSK3.

Human NKR-P1 (CD161, KLRB1 ) and its ligand LLT1 ( CLEC2D ) are a prototypical inhibitory C-type lectin-like receptor:ligand pair of NK cells with a critical role in homing lymphocytes to immune-privileged sites, particularly in multiple sclerosis, rheumatoid arthritis, and Crohn’s disease. Furthermore, NKR-P1:LLT1 inhibitory signaling is associated with glioblastoma, non-Hodgkin’s lymphoma, breast, and prostate cancer. However, the lack of structural data on the formation of the NKR-P1:LLT1 complex limits our understanding of this signaling. We thus solved the crystal structures of NKR-P1 and the NKR-P1:LLT1 complex. NKR-P1 forms a homodimer with an unexpected arrangement that enables LLT1 binding in two modes, bridging two LLT1 molecules, thereby forming interaction clusters suggestive of an inhibitory immune synapse. Moreover, observing the formation of these clusters by SEC-SAXS analysis in solution and by dSTORM super-resolution microscopy on the cell surface, and following their role in receptor signaling using in vitro cytotoxicity assay with freshly isolated NK cells, we show how NKR-P1:LLT1 clustering allows these proteins to overcome the weak affinity of C-type lectin-like receptors to their ligands. Furthermore, only the ligation of both primary and secondary LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. Therefore, our findings show how inhibitory receptor cross-linking and clustering work together to trigger signal transduction upon cellular contact in the immune synapse.

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity‐dependent biotin identification (BioID)‐based proteomics approach, using the established IMC marker protein Photosensitized INA‐Labelled protein 1 (PhIL1) as bait in asexual blood‐stage parasites. Subsequent mass spectrometry‐based peptide identification revealed enrichment of twelve known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP‐tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represent structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood‐stage parasites.

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of twelve known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represent structures described as apical annuli in Toxoplasma gondii . Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites.