30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

Understanding function and assembly of the atypical centrosome in Plasmodium falciparum

Abstract text Throughout its life cycle Plasmodium falciparum undergoes several stages of extensive proliferation that are integral to survival and transmission of this malaria-causing parasite. Rapid parasite multiplication in the human red blood cell occurs via an atypical cell division mode, called schizogony, and relies on a particularly divergent centrosome. This so called centriolar plaque, functions as the main microtubule organizing center (MTOC) for the mitotic spindle and lacks conventional structures, such as centrioles. Our previous investigation using a combination of super-resolution, live cell, and correlative microscopy have revealed that it rather consists of two amorphous protein dense regions spanning across the nuclear membrane (see Figure). Composition and assembly of this atypical centrosome remain largely unknown. To better understand the centriolar plaque we investigated centrins, which are small calcium-binding proteins and among the very few universally conserved MTOC components. Contrary to previous hypotheses we found liquid-liquid phase-separation (LLPS) as a driver behind the calcium-inducible self-assembly of some centrins in vitro. LLPS has emerged as biophysical model explaining the accumulation of concentrated proteins in droplet-like structures, which can contribute to the formation of membraneless compartments, such as centrosomes. To verify LLPS of PfCentrin1 in parasites we employed live cell STED and designed a novel inducible overexpression system. Co-immunoprecipitation of PfCentrin1 further revealed a Sfi1-like protein (PfSlp) as novel centriolar plaque component. Conditional knock down of PfSlp caused a growth delay in blood stages and a reduced number of daughter cells. Surprisingly, intranuclear tubulin abundance was significantly increased, which raises the hypothesis that the centriolar plaque might be implicated in regulating tubulin homeostasis. Time-lapse microscopy revealed that this misregulation prevented or delayed spindle extension. Taken together our data indicates that the centrin/Slp protein pair is a core component of the centriolar plaque and might implicate phase separation in its assembly.

Molecular Parasitology

Parasite-Host Interaction

Talk

Investigating the mechanism of heterogeneous protein accumulation among the nuclei of a Plasmodium falciparum schizont

Abstract text Plasmodium falciparum proliferates through schizogony in the clinically relevant blood stage of infection. During schizogony, the nuclei multiply several times and the parasite develops into a multinucleated stage before cellularization occurs and daughter cells are released. To facilitate rapid parasite proliferation, the nuclei multiply asynchronously despite residing in the same cytoplasm. Moreover, the DNA replication fork protein P. falciparum PCNA1 accumulates only in those nuclei that replicate their DNA. However, the molecular mechanisms that enable heterogeneous nuclear accumulation of P. falciparum PCNA1 and thus asynchronous nuclear multiplication are not yet understood. To investigate this phenomenon, we used super-resolution live-cell microscopy. Our data suggest that P. falciparum PCNA1 initially accumulates close to the centrosome before relocating towards the nuclear periphery. This may indicate that the progression of the S-phase follows a conserved intranuclear pattern and that PCNA1 accumulation in subnuclear compartments underlies a specific spatiotemporal control. To elucidate this regulation, we are using mutagenesis and synthetic approaches to analyse how different protein motifs of P. falciparum PCNA1 contribute to its heterogeneous accumulation. Our data suggest that several amino acids play an important role. This work also identified a poorly conserved P. falciparum PCNA1 lysine-rich motif that was sufficient to accumulate GFP at the centrosomes. Building on these findings, we are now testing the behaviour of additional mutants and profiling the interactomes of wild-type and mutant P. falciparum PCNA1. Together, this will grant further molecular insight into the underlying mechanism of heterogeneous protein accumulation among P. falciparum nuclei.

Drug Development/Target Identification

Prof.

The histone methyltransferase PfSET2 regulates gene expression crucial for gametocyte development

Abstract text The intracellular and extracellular survival of the malaria parasite Plasmodium falciparum in the human and mosquito hosts is dependent on rapid morphological and physiological changes, which are coordinated by various gene regulation mechanisms. Growing evidence suggests the contribution of epigenetic control mechanisms, in particular histone post-translational modifications, during intraerythrocytic replication and immune evasion of the asexual blood stage parasites. In contrast, the role of histone post-translational modifications during the sexual development of the parasite is not well studied. Previously conducted chemical loss-of-function studies by Ngwa et al. (2019), using the histone methyltransferase inhibitor BIX-01294, reveiled significant changes in the gene expression pattern of gametocytes and an impairment in gametocyte development and gametogenesis. In this study, we aimed to investigate the role of the histone methyltransferase PfSET2, one of ten known SET proteins of P. falciparum, in gene regulation during gametocyte development. We show that PfSET2 is expressed in the asexual and sexual blood stages. PfSET2 gene disruption results in changes in the histone methylation and gene expression patterns of the asexual blood stages and in the abrogation of gametocyte development. Our data point to a pivotal function of PfSET2 in the epigenetic regulation of gene expression crucial for gametocyte vitality.

How a patatin-like phospholipase rules mitochondrial function in malaria parasites

Abstract text Introduction: Plasmodium parasites rely on a functional electron transport chain (ETC) within their mitochondrion for proliferation, and compounds targeting mitochondrial functions are validated antimalarials. In other organisms, mitochondrial functions have been shown to be heavily influenced by the phospholipid composition of mitochondrial membranes. However, the importance of phospholipid-modifying enzymes for mitochondrial function in Plasmodium parasites has not been analyzed so far. Objectives: In this study, we aim to characterize the physiological function of the Plasmodium falciparum patatin-like phospholipase 2 (PfPNPLA2, PF3D7_1358000) during asexual and sexual development of the parasite. Materials &amp; methods: We endogenously tagged PfPNPLA2 with green fluorescent protein and generated PfPNPLA2-deficient parasites using reverse genetics. Subsequently, we analyzed transgenic parasites using fluorescence microscopy and various cellular assays. Results: We show that PfPNPLA2 resides in the mitochondrion and its experimental deletion impairs asexual replication. The physiological consequences of this PfPNPLA2 deletion are i) hypersensitivity to proguanil and inhibitors of the mitochondrial ETC including atovaquone, ii) lower mitochondrial respiration measured by quantification of oxygen consumption rates, and iii) a reduced mitochondrial membrane potential. Subsequent lipidomics-based analysis of the mitochondrial phospholipid cardiolipin (CL) reveals that deletion of PfPNPLA2 is associated with an increase of CL with shorter and more saturated acyl-groups, suggesting a potential role of PfPNLA2 in CL remodelling. We finally show that PfPNPLA2-deficient parasites also display profound defects in gametocytogenesis with most parasites only developing to stage III gametocytes, underlining the importance of a functional mitochondrial ETC for sexual development of the parasite. Conclusion: PfPNPLA2 has a key role for mitochondrial function that is important for asexual replication and sexual development of the parasite.

Expansion of a malarial parasite antigen family in the mammalian host can be explained by synthetic viability of a lethal Plasmodium berghei cysteine protease defect

Abstract text During life cycle progression of malarial parasites, efficient and timely Plasmodium exit events from replication-permissive compartments are central steps in parasite stage conversion. Papain-like cysteine proteases of a family of Plasmodium-specific serine repeat antigens (SERAs) are central mediators of parasite egress and conserved across the genus Plasmodium. One member, termed SERA3 in the murine malaria model, is likely essential during blood infection. In this study, we employed a systematic reverse genetics approach to characterize the function of P. berghei SERA3. As predicted, we failed to generate P. berghei parasites lacking SERA3 by targeted gene deletion. Surprisingly, in the presence of additional gene deletions, i.e. deletion of both active site serine (SERAser) members of the protease family, SERA1 and 2, parasites were viable and progressed through the parasite life cycle. Systematic phenotyping of sera1-3(-) parasites revealed that infected mice fail to develop signature symptoms of cerebral malaria and are able to clear an infection. Importantly, parasite clearance cannot be attributed to reduced virulence, since it only occurs when initiated by sporozoite infection, but not via transfusion of sera1-3(-) blood stages. Parasite clearance is strictly B- and T-cell dependent and elicits high titres of anti-parasite antibodies for up to one year. Intriguingly, the sera1-3 triple knockout is a genocopy of the avian malarial parasite SERA repertoire. Apparently, SERA3 dysfunction is incompatible with asexual parasite growth in the presence of functional SERAser proteins. This suggests that the up to six SERAser protease family members could only evolve after fixation of SERA3 in the mammalian genomes. This genetic observation, known as synthetic viability, leads us to propose that expansion of the SERA gene family was vital to establish chronic and repeated Plasmodium infections in the mammalian host.

Session I
Molecular Parasitology I – Protozoa 1 (Plasmodium)

Termin

Vorsitz

Programm

Session IMolecular Parasitology I – Protozoa 1 (Plasmodium)

Termin

Vorsitz

Programm

Session I
Molecular Parasitology I – Protozoa 1 (Plasmodium)