Biology subjects

Passecker, A.

Publications and source records attributed to Passecker, A..

3 recordsLinked to original sources

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↗