Biology subjects

Brancucci, N. M. B.

Publications and source records attributed to Brancucci, N. M. B..

11 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↗

bioRxiv2026

Multidimensional in vitro assay for antimalarial combination testing and pharmacodynamic modeling - the MULT-i2 assay

Malaria remains a major global health challenge, with emerging partial resistance to first-line therapies in Africa threatening current control efforts. Drug combinations are essential to improve treatment efficacy and restrain resistance development. However, in vitro assays that quantify parasite viability after drug exposure and characterize pharmacodynamic drug interactions are labor- and resource-intensive, with standard approaches such as the parasite reduction ratio assay limiting systematic, high-resolution evaluation of drug combinations. We present the MUltidimensional Luminescence Test for integration of interactions (MULT-i2), an in vitro assay that enables scalable, high-resolution assessment of parasite viability across multidimensional drug concentration spaces. For dual drug combinations, the MULT-i2 assay characterizes interaction surfaces while requiring [~]50-fold fewer resources and more than two-fold less time than conventional methods, enabling exploration of broader combination scenarios. The assay combines a highly sensitive chemiluminescence readout with inducible reporter expression in Plasmodium falciparum, supporting potential extension to multidimensional combination testing. Using the general pharmacodynamic interaction (GPDI) model, the MULT-i2 assay quantified interaction potency and directionality, confirming and refining the known synergy between atovaquone and proguanil, and revealing detailed interaction patterns for additional drug combinations. Overall, this approach provides an efficient framework for testing and characterizing pharmacodynamic drug interactions and supports the rational development of antimalarial combination therapies.

microbiology↗

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↗

bioRxiv2024

The three Plasmodium falciparum Aurora-related kinases display distinct temporal and spatial associations with mitotic structures in asexual blood stage parasites and gametocytes

Aurora kinases are crucial regulators of mitotic cell cycle progression in eukaryotes. The protozoan malaria parasite Plasmodium falciparum replicates via schizogony, a specialised mode of cell division characterized by consecutive asynchronous rounds of nuclear division by closed mitosis followed by a single cytokinesis event producing dozens of daughter cells. P. falciparum encodes three Aurora-related kinases (PfARKs) that have been reported essential for parasite proliferation, but their roles in regulating schizogony have not yet been explored in great detail. Here, we engineered transgenic parasite lines expressing GFP-tagged PfARK1-3 to provide a systematic analysis of their expression timing and subcellular localization throughout schizogony as well as in the non-dividing gametocyte stages, which are essential for malaria transmission. We demonstrate that all three PfARKs display distinct and highly specific and exclusive spatiotemporal associations with the mitotic machinery. In gametocytes, PfARK3 is undetectable and PfARK1 and PfARK2 show male-specific expression in late stage gametocytes, consistent with their requirement for endomitosis during male gametogenesis in the mosquito vector. Our combined data suggest that PfARK1 and PfARK2 have non-overlapping roles in centriolar plaque maturation, assembly of the mitotic spindle, kinetochore-spindle attachment and chromosome segregation, while PfARK3 seems to be exquisitely involved in daughter cell cytoskeleton assembly and cytokinesis. These important new insights provide a reliable foundation for future research aiming at the functional investigation of these divergent and possibly drug targetable Aurora-related kinases in mitotic cell division of Plasmodium falciparum and related apicomplexan parasites.

cell biology↗

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

From contigs to chromosomes: automatic Improvement of Long Read Assemblies (ILRA)

Recent advances in long read technologies not only enable large consortia to aim to sequence all eukaryotes on Earth, but they also allow individual laboratories to sequence their species of interest with relatively low investment. Although there is a promise of long read technologies to obtain "perfect genomes", the number of contigs often exceeds the number of chromosomes by far, containing many insertion and deletion errors around homopolymer tracks. To overcome these issues, we implemented the ILRA pipeline to correct long read-based assemblies, so contigs are reordered, renamed, merged, circularized, or filtered if erroneous or contaminated, and Illumina reads are used to correct homopolymer errors. We successfully tested our approach by improving the genomes of Trypanosoma brucei and Leptosphaeria spp, and generated four novel Plasmodium falciparum assemblies from field samples. We found that correcting homopolymer tracks reduced the number of genes incorrectly annotated as pseudogenes, but an iterative correction seems to be required to correct larger numbers of sequencing errors. In summary, we described and compared the performance of our new tool, which improved the quality of novel long read assemblies of genomes up to 1Gbp. AvailabilityThe tool is available at GitHub: https://github.com/ThomasDOtto/ILRA.

bioinformatics↗

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↗

bioRxiv2019

3D imaging of undissected optically cleared Anopheles stephensi mosquitoes infected with Plasmodium parasites

Malaria is a life-threatening disease, caused by Apicomplexan parasites of the Plasmodium genus. The Anopheles mosquito is necessary for the sexual replication of these parasites and for their transmission to vertebrate hosts, including humans. Imaging of the parasite within the insect vector has been attempted using multiple microscopy methods, most of which are hampered by the presence of the light scattering opaque cuticle of the mosquito. So far, imaging of the Plasmodium mosquito stages depended on either sectioning or surgical dissection of important anatomical sites, such as the midgut and the salivary glands. Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) enable imaging fields of view in the centimeter scale whilst providing micrometer resolution. In this paper, we present reconstructions of the whole body of Plasmodium-infected, optically cleared Anopheles stephensi mosquitoes and their midguts. The 3D-reconstructions from OPT imaging show detailed features of the mosquito anatomy and enable overall localization of parasites in midguts. Additionally, LSFM imaging of mosquito midguts shows detailed distribution of oocysts in extracted midguts.

microbiology↗