Biology subjects

Passecker, A.

Publications and source records attributed to Passecker, A..

7 recordsLinked to original sources

bioRxiv2026

A high-throughput compound screen identifies multiple druggable targets in Plasmodium falciparum transmission stages

Most antimalarials are ineffective against the sexual transmission stages, known as gametocytes, of the malaria parasite Plasmodium falciparum. Their low sensitivity to drugs is attributed to limited compound uptake and a poorly understood form of cellular quiescence. Our current understanding of druggable transmission-blocking processes is therefore limited. Based on genetically engineered parasites that facilitate the mass production of synchronous mature gametocytes, we developed a high throughput drug screening platform that allowed us to test more than 50,000 compounds for gametocytocidal effects in one day. By screening a diversity-oriented library, we identified over 40 molecules that kill mature gametocytes in the low nanomolar range. Using resistance selection coupled to whole genome sequencing and drug-target interaction modelling, we followed up on three chemically tractable compounds that are also highly active against asexual parasites and prevent gametocyte transmission to mosquitoes. We show that the compound ONX-0914, a specific inhibitor of the {beta}5i/LMP7 subunit of human immunoproteasomes, targets the parasite proteasomal {beta}5 subunit. In contrast, the compounds CR-1-31-B and brusatol interfere with translation by targeting eukaryotic initiation factor 4A (eIF4A) and the peptidyl transferase center (PTC) of the 80S ribosome, respectively. Interestingly, parasite resistance to brusatol, a broad-spectrum antitumor drug, is linked to the differential modification of specific rRNA bases near the ribosomal A-site, mediated by altered base specificity of a rRNA methyltransferase. In summary, we successfully combined high-throughput compound screening with drug target deconvolution to reveal the targets and mode-of-action for three potent gametocytocidal molecules and discover the mechanism of resistance to the anti-tumorigenic drug brusatol. In addition to critically advancing our understanding of mature gametocyte biology and druggable processes in P. falciparum transmission stages, our observations made with brusatol-resistant parasites may become relevant for anti-cancer drug research.

molecular biology↗

bioRxiv2024

An all-in-one pipeline for the in vitro discovery and in vivo testing of Plasmodium falciparum malaria transmission blocking drugs

Elimination and eradication of malaria will depend on new drugs with potent activity against Plasmodium falciparum mature stage V gametocytes, the only stages able to infect the mosquito vector for onward parasite transmission. The identification of molecules active against these quiescent stages is difficult due to the specific biology of gametocyte maturation and challenges linked to their cultivation in vitro. Furthermore, the antimalarial drug development pipeline lacks a suitable animal model for evaluating the transmission-blocking potential of promising lead compounds and preclinical and clinical drug candidates in vivo. Here, we established a transmission-blocking drug discovery and development platform based on transgenic P. falciparum parasites engineered to produce large numbers of pure stage V gametocytes expressing a red-shifted firefly luciferase as reporter for cellular viability. This NF54/iGP1_RE9Hulg8 line facilitated the development of a highly efficient and robust in vitro screening assay for the identification of stage V gametocytocidal compounds. Importantly, by infecting humanized NODscidIL2R{gamma}null mice with pure NF54/iGP1_RE9Hulg8 stage V gametocytes, we also established a preclinical P. falciparum in vivo transmission model. Using whole animal bioluminescence imaging and quantification of gametocyte densities over a period of 14 days, we assessed the gametocyte killing and clearance kinetics in vivo of antimalarial reference drugs as well as five clinical drug candidates and identified markedly different pharmacodynamic response profiles. Furthermore, we successfully integrated this mouse model with mosquito feeding assays and thus firmly established a valuable tool for the systematic in vivo evaluation of gametocytocidal and transmission-blocking drug efficacy. One sentence summaryWe applied robust new assays for gametocytocidal drug discovery and in vivo efficacy testing using a humanized mouse model for malaria transmission

microbiology↗

bioRxiv2022

FKBP35 secures ribosome homeostasis in Plasmodium falciparum

Plasmodium falciparum accounts for the majority of over 600000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with alternative modes of action is thus undoubted. The FK506-binding protein PfFKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus). Whilst there is considerable interest in targeting PfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing and its function in parasite biology remains elusive. Here, we show that limiting PfFKBP35 levels are lethal to P. falciparum and result in a delayed death-like phenotype that is characterized by defective ribosome homeostasis and stalled protein synthesis. Our data furthermore suggest that FK506, unlike the action of this drug in model organisms, exerts its anti-proliferative activity in a PfFKBP35-independent manner and, using cellular thermal shift assays, we identify putative FK506-targets beyond PfFKBP35. In addition to revealing first insights into the function of PfFKBP35, our results show that FKBP-binding drugs can adopt non-canonical modes of action - with major implications for the development of FK506-derived molecules active against Plasmodium parasites and other eukaryotic pathogens.

molecular biology↗

bioRxiv2021

Revisiting the effect of pharmaceuticals on transmission stage formation in the malaria parasite Plasmodium falciparum

Malaria parasites rely on specialized stages, called gametocytes, to ensure human-to-human transmission. The formation of these sexual precursor cells is initiated by commitment of blood stage parasites to the sexual differentiation pathway. Plasmodium falciparum, the most virulent of six parasite species infecting humans, employs nutrient sensing to control the rate at which sexual commitment is initiated, and the presence of stress-inducing factors, including antimalarial drugs, has been linked to increased gametocyte production in vitro and in vivo. These observations suggest that therapeutic interventions may promote gametocytogenesis and malaria transmission. Here, we engineered a P. falciparum reporter line to quantify sexual commitment rates after exposure to antimalarials and other pharmaceuticals commonly prescribed in malaria-endemic regions. Our data reveal that some of the tested drugs indeed have the capacity to elevate sexual commitment rates in vitro. Importantly, however, these effects are only observed at drug concentrations that inhibit parasite survival and only rarely result in a net increase of gametocyte production. Using a drug-resistant parasite reporter line, we further show that the gametocytogenesis-promoting effect of drugs is linked to general stress responses rather than to compound-specific activities. Altogether, we provide conclusive evidence for the absence of mechanistic links between the regulation of sexual commitment and the activity of commonly used pharmaceuticals in vitro. Our data hence contradict scenarios in which therapeutic interventions would promote the spread of drug-resistant parasites or malaria transmission in general.

cell biology↗

bioRxiv2021

The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites

Cyclic AMP (cAMP) signalling is crucial for the propagation of asexual malaria blood stage parasites. Recent work on Plasmodium falciparum demonstrated that phosphorylation of the invasion ligand AMA1 by the catalytic subunit of cAMP-dependent protein kinase A (PfPKAc) is an essential step during parasite invasion into red blood cells. However, the exact mechanisms regulating PfPKAc activity are only partially understood and PfPKAc function has not been extensively studied in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown mutant, we confirm the essential role for PfPKAc in erythrocyte invasion and demonstrate that PfPKAc is involved in regulating gametocyte deformability. Interestingly, we observed that the conditional overexpression of PfPKAc also caused a profound lethal phenotype by preventing intra-erythrocytic parasite multiplication. Whole genome sequencing of parasites selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the putative parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we show that PfPDK1 is essential for PfPKAc activation, most likely by phosphorylating T189 in the PfPKAc activation loop. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and identify PfPDK1 as a crucial upstream regulator in this pathway and potential new drug target.

microbiology↗

bioRxiv2020

Investigation of heterochromatin protein 1 function in the malaria parasite Plasmodium falciparum using a conditional domain deletion and swapping approach

The human malaria parasite Plasmodium falciparum encodes a single ortholog of heterochromatin protein 1 (PfHP1) that plays a crucial role in the epigenetic regulation of various survival-related processes. PfHP1 is essential for parasite proliferation and the heritable silencing of genes linked to antigenic variation, host cell invasion and sexual conversion. Here, we employed CRISPR/Cas9-mediated genome editing combined with the DiCre/LoxP system to investigate how the PfHP1 chromodomain (CD), hinge domain and chromoshadow domain (CSD) contribute to overall PfHP1 function. We show that the C-terminal 76 residues are responsible for targeting PfHP1 to the nucleus. Furthermore, we reveal that each of the three functional domains of PfHP1 are required for heterochromatin formation, gene silencing and mitotic parasite proliferation. Finally, we discovered that the hinge and CSD domains of HP1 are functionally conserved between P. falciparum and P. berghei, a related malaria parasite infecting rodents. In summary, our study provides new insights into PfHP1 function and offers a tool for further studies on epigenetic regulation and life cycle decision in malaria parasites.

microbiology↗

bioRxiv2020

CRISPR/Cas9-engineered inducible gametocyte producer lines as a novel tool for basic and applied research on Plasmodium falciparum malaria transmission stages

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission to other human hosts via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialized cells. Here, we engineered marker-free P. falciparum inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes. Through targeted overexpression of the sexual commitment factor GDV1, iGP lines consistently achieve sexual commitment rates of 75% and produce gametocytes that are infectious to mosquitoes. Subsequent tagging of a nucleoporin allowed us to visualize marked nuclear transformations during gametocytogenesis and demonstrates that further genetic engineering of iGP lines is an invaluable tool for the targeted exploration of gametocyte biology. We believe the iGP approach developed here opens up unprecedented opportunities that will expedite future basic and applied research on P. falciparum transmission stages.

microbiology↗