Biology subjects

Voss, T. S.

Publications and source records attributed to Voss, T. S..

6 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

The ApiAP2 factor PfAP2-HC is an integral component of heterochromatin in the malaria parasite Plasmodium falciparum

Malaria parasites undergo a highly complex life cycle in the human host and the mosquito vector. The ApiAP2 family of sequence-specific DNA-binding proteins plays a dominant role in parasite development and life cycle progression. Of the ApiAP2 factors studied to date, most act as transcription factors regulating stage-specific gene expression. Here, we characterised a new ApiAP2 factor in Plasmodium falciparum (PF3D7_1456000) that we termed PfAP2-HC. Via detailed investigation of several single or double genetically engineered parasite lines, we demonstrate that PfAP2-HC specifically binds to heterochromatin throughout the genome. Intriguingly, PfAP2-HC does not bind DNA in vivo and recruitment of PfAP2-HC to heterochromatin is independent of its DNA-binding domain but strictly dependent on heterochromatin protein 1. Furthermore, our results suggest that PfAP2-HC functions neither in the regulation of gene expression nor in heterochromatin formation or maintenance. In summary, our findings reveal that PfAP2-HC constitutes a core component of heterochromatin in malaria parasites. They furthermore identify unexpected properties of ApiAP2 factors and suggest substantial functional divergence among the members of this important family of regulatory proteins.

microbiology↗

bioRxiv2020

GDV1 C-terminal truncation of 39 amino acids disrupts sexual commitment in Plasmodium falciparum

Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasites life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase KO lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but both lines harbour the same mutation that leads to a truncation in the extreme C-terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line we show that the truncation in the GDV1 C-terminus does neither interfere with the nuclear import of GDV1 nor its interaction with HP1 in vitro, but appears important to sustain GDV1 protein levels and thereby sexual commitment. ImportanceTransmission of malaria causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood, to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the lifecycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C-terminus of the Gametocyte Development 1 protein (GDV1), which abrogates sexual development. The mutation destabilises the protein but not its interaction with its cognate binding partner HP1. This suggest an important role for the GDV1 C-terminus beyond trafficking and protein stability.

microbiology↗

bioRxiv2020

Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.

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↗