BioMalPar XIX - biology and pathology of the malaria parasite: Day 3
MESA Correspondents bring you cutting-edge coverage from the BioMalPar XIX: biology and pathology of the malaria parasite
Day 3: Thursday, 25th May 2023
Scientific Session 5 – Systems Biology of Malaria: Molecules, Cells, Physiology and Pathobiology
Elisabeth Egan (Stanford University, United States) spoke about the significance of the red blood cell (RBC) receptor, CD44 during P. falciparum invasion and the importance of implementing an improved screening approach to identify key host membrane receptors for malaria. Using nucleated human hematopoietic stem cells (HSPCs), Egan’s group developed forward genetic screens of differentiated HSPCs tagged with green fluorescence protein (GFP) plasmids to identify host factors related to P. falciparum invasion. CD44 and CD55 were identified as essential for host cell invasion. CRISPR/Cas9 technology was used to further investigate the role of CD44s in RBCs. CD44-null cultured RBCs (cRBCs) obtained from primary HSPCs were compared to isogenic wild-type cRBCs. The results revealed a greater reduction in P. falciparum invasion in the CD44-null cRBCs compared to the wild type. Further exploration uncovered that ligands EBA175 and EBA140 from the parasite interacted with the CD44 receptor. Additionally, monoclonal antibodies raised against CD44 including BRIC 222, BRIC 170, BRIC 235, KZ1, and IM7 were successfully used to promote P. falciparum invasion. This effect was due to CD44 cross-linking, which in turn increased basigin detection on the RBC surface. Overall, Egan et al. discovered that both BRIC 222 and EBA175 induced changes in the RBC membrane, which increased basigin accessibility and promoted invasion. These findings enhance the understanding of the mechanisms behind P. falciparum infections and suggest potential targets for future therapeutic interventions.
Brendan Farrell (University of Oxford, United Kingdom) highlighted the importance of the PfPCRCR complex (formed by PfPTRAMP, PfCSS, PfRIPR, PfCyRPA and PfRH5) in the interaction between merozoites and basigin on erythrocytes. Cryo-EM analysis of this complex revealed how PfCyRPA, PfRIPR, and PfRH5 proteins interact at a molecular level. For instance, PfRH5 and PfRCR were found to be binding with equal affinity to basigin and the former showed no significant conformational changes during invasion. Surprisingly, PfRH5 possessed a crucial disulfide bond that enabled PfRH5 to act as a pore. The study also determined that the elongated tail of PfRIPR interacted with the PfCSS-PfPTRAMP complex in merozoites. Finally, antibodies targeting the basigin-PfRH5 binding disrupted the PfPCRCR complex's bridging function, hindering invasion. Farrell concluded by stating that because PfPCRCR bridges the parasite and erythrocytes, these findings could contribute to rational vaccine design for malaria prevention.
Tim Satchwell (University of Bristol, United Kingdom) discussed the role of PfRH5-basigin in mediating invasion and elucidated the properties and traits of basigin that make invasion possible. Using in vitro erythropoiesis and a CRISPR/Cas9 approach, Satchwell investigated the role of uncoupled MCT1-basigin interaction in RBCs during parasite invasion. Through genetic manipulation, reticulocytes with uncoupled basigin and reduced MCT1 expression were created. The knockout of basigin led to an 80% reduction in MCT1 expression. However, reintroducing basigin mutants lacking the transmembrane helix responsible for MCT1 interaction, allowed the surface presentation of the extracellular domain for binding with PfRH5. This facilitated merozoite invasion independent of MCT1, suggesting that the interaction between basigin and MCT1 may not be essential for the invasion process. Overall, Satchwell's presentation provided valuable insights into the functional significance of MCT1-basigin in the host cell membrane context, ultimately contributing to the understanding of the invasion mechanism.
Anna Truong (Duke University, United States) began the talk by emphasizing the significance of ubiquitination in P. falciparum, specifically focusing on the gene PfUbc13. Ubiquitin, an 8-kDa protein, acts as a signaling molecule that attaches to substrate proteins through enzymatic reactions involving E1, E2, and E3 enzymes. The study aimed to uncover PfUbc13 interacting partners, decipher their regulatory mechanisms and identify target substrate proteins associated with PfUbc13. Initial studies showed that deletion of PfUbc13 amplified the sensitivity of the parasite to the antimalarial drug dihydroartemisinin, alluding to its involvement in drug activity. To assess PfUbc13 activity, a chemical probe was employed, and downstream target proteins were identified using mass spectrometry-based proteomic techniques. With this analysis 64 potential substrate proteins of PfUbc13 were successfully identified. Subsequently, gene ontology enrichment revealed that these proteins were clustered into four major areas: metabolic processes, regulation of biological processes, intracellular transport, and protein organization. This research offers valuable insights into the interactions of PfUbc13 and the mechanism of antimalarial drugs thus contributing to the ongoing efforts in malaria drug discovery.
Rita Tewari’s (University of Nottingham, United Kingdom) presentation began with a brief introduction to the diverse cell division mechanism in Plasmodium. She addressed the significance of atypical parasitic cell division and its implications for parasite survival. Plasmodium exhibits asynchronous division, including two modes of mitosis (closed and end mitosis) and a brief meiosis phase. Aurora related kinases and microtubule associated proteins were identified as major regulatory molecules governing cell division in P. berghei. Tewari's research focused on defining the functions of ARK2 in the centrosomal complex microtubule-organizing center (MTOC) during P. berghei's schizogeny. ARK2, associated with scaffold proteins, was found to colocalize with the spindle, displaying dynamics similar to Ndc80 suggesting that it has a polar distribution in the nuclear compartment. During male gametogenesis, ARK2 influenced early oocyte formation, leading to exflagellation. Additionally, Tewari and colleagues found that ARK2 was associated with EB1 at the centrosome, which had similar spindle dynamics as Ncd80, exhibiting a comparable profile. The Ark2-EB1 association was found to have a role in endomitosis during oocyst development. Tewari ended her presentation by mentioning the need for further research exploring the function of ARK2-EB1 interactions to understand spindle dynamics and spatial organisation during cell division in Plasmodium.
Amit Kumar Subudhi’s (King Abdullah University of Science and Technology, Saudi Arabia) presentation focused on the crucial role of PfAP2-P, an Apicomplexan AP2 transcription factor, in malaria pathogenicity. The study highlighted PfAP2-P's involvement in var gene regulation, merozoite development, and parasite egress during the intraerythrocytic developmental cycle. ChIP-seq experiments revealed PfAP2-P's binding to gene promoters of other AP2 proteins, controlling antigenic variation and host cell remodeling. Deletion of PfAP2-P resulted in derepressed var gene expression and overexpression of early gametocyte markers, indicating its role in sexual stage conversion. Chromosome conformation capture experiments showed reduced chromatin interactions upon PfAP2-P deletion. Overall, the findings emphasized PfAP2-P as a vital upstream regulator governing key pathogenic processes in distinct parasite developmental stages.
Stuart Ralph’s (University of Melbourne, Australia) presentation was focused on the significance of the Kelch 13 (K13) protein. K13, which has been associated with artemisinin resistance, seems to be important for the formation of cytostomes, which are invagination structures involved in haemoglobin uptake by P. falciparum. Specifically, Ralph revealed that K13 stabilizes the cytostome by forming doughnut-shaped rings around the cytostomal neck, consistent with electron microscopy findings. The absence of K13 resulted in abnormal cytostomal structures as shown by electron tomography and serial-block-face SEM. Additionally, fractionation experiments indicated reduced haemoglobin uptake, haem release, and artemisinin activation in K13 mutants, leading to lower haem and hemozoin production. In addition, a connection between K13 and ubiquitin signalling was suggested but requires further investigation. Overall, these findings shed light on the crucial role of K13 in cytostome formation and its link to artemisinin resistance, aiding the development of strategies to combat drug-resistant malaria.
Scientific Session 6 – New Topics and Tools for Malaria Research and Future Innovation
Maria Bernabeu’s (European Molecular Biology Laboratory, Barcelona, Spain) talk focused on the use of a three-dimensional blood-brain barrier (3D-BBB) in vitro model to study malaria pathogenesis. The model consists of a microfluidic network within a collagen hydrogel, simulating various flow conditions in the brain vasculature. Two models were developed consisting of either primary human brain microvascular endothelial cells or induced pluripotent stem cell-derived endothelial cells (iPSC-EC) in combination with astrocytes and pericytes. The iPSC-EC model demonstrates improved barrier properties, such as physiological permeability rate and enhanced expression of tight junction markers. With these models, Bernabeu’s team aims to identify host and parasite factors driving cerebral malaria and further explore the underlying molecular mechanisms. Consequently, 3D-BBB models were exposed to different P. falciparum stages or parasite product media, which disrupted the endothelial cells of the 3D-BBB. Through various molecular techniques, changes in barrier integrity and the impact on BBB cell composition were examined. Additionally, scRNAseq revealed that BBB cells have a unique transcriptional profile after exposure to parasite toxins which altered signaling pathways in endothelial cells, astrocytes, and pericytes. Overall, the 3D-BBB model uncovers novel insights into the molecular mechanisms of cerebral malaria and could potentially contribute to the development of therapies to reduce patient mortality.
Alexandra Probst (Novartis Institute for Tropical Diseases, United States) discussed genetic modification of P. cynomolgi as a model to gain insights into targeting P. vivax parasites at the liver hypnozoite stage. First, using CRISPR/Cas9, Probst’s team replaced the endogenous circumsporozoite protein (CSP) gene, which is essential for parasite development in the mosquito and for liver infection, with PvCSP from P. vivax. The transgenic parasites completed their life cycle and generated infectious sporozoites, which could cause relapsing infections in monkeys. Then, to pursue drug discovery purposes, transgenic parasites expressing HaloTag at the host-parasite interface during the liver stage were generated to disrupt the host ubiquitin-proteasome system. The HaloTag was bound to bifunctional molecules potentially targeting hypnozoites for degradation. In addition, Probst highlighted the potential of employing transgenic lines for various applications in malaria research, such as live fluorescence microscopy, proteomics, and protein degradation studies. Furthermore, Probst mentioned the prospect of utilizing positron emission tomography (PET) for in vivo imaging of infected monkeys, enabling the monitoring of liver burden during malaria infection. This research offers potential strategies to target hypnozoites and combat relapsing P. vivax malaria infections.
Yunuen Avalos Padilla (Institute for Bioengineering of Catalonia, Spain) started by presenting the endosomal sorting complex required for transport (ESCRT), and in particular, the ESCRT-III sub-complex which is involved in extracellular vesicles (EVs) formation. Even though RBCs lost the majority of their organelles, they still produce EVs with an alternative pathway involving PfBro1, PfVps32 and PfVps60 proteins. By using antibodies raised against PfVps32, a reduction of parasite viability was observed. To overcome the cost limitations of antibodies, the study used aptamers as an alternative approach. Aptamers specific to PfVps32 and the AAA-ATPase PfVps4 were isolated and were shown to bind to Plasmodium-infected RBCs. Furthermore, fluorescence microscopy confirmed the binding of the aptamers to parasites and vesicles present on the surface of the host. The aptamer targeting PfVps32 exhibited the lowest IC50 value in inhibiting parasite growth. Despite its low level, it demonstrated its potential as an antiparasitic agent. Lastly, Avalos-Padilla discussed future plans, which included analyzing the entire pool of aptamers and determining their IC50 values through next-generation sequencing (NGS). Additionally, efforts will be made to enhance aptamer stability and reduce degradation by formulating them into polymersomes, potentially as carriers for other antimalarial drugs. The study highlighted the potential of aptamers as an efficient and cost-effective tool for targeting P. falciparum.
Nedal Darif (European Molecular Biology Laboratory, Heidelberg, Germany) spoke about oocyst formation and the use of advanced imaging techniques such as volume electron microscopy (vEM) as a proof of concept to differentiate the stages of oocyst biogenesis. Darif proposed using a combination of vEM, Synchrotron X-Ray Tomography (SXCT), confocal targeting, and electron microscopy to capture high-resolution images of oocysts and sporozoites. In the presentation, Darif detailed images identifying the location of oocytes at various developmental stages. By utilizing a blend of targeted imagery and pre-trained neural networks, the team achieved the remarkable feat of predicting and recreating the parasite's developmental process. This innovative approach also led to the identification of novel structures, including an electron-dense tubular organelle within the sporoblast, which presents an avenue for further investigation. Additionally, Darif discussed the ongoing efforts to construct a comprehensive cell atlas of oocysts and sporozoites, employing advanced imaging techniques to unveil the intricate stages of Plasmodium development.
Jacquin Niles (Massachusetts Institute of Technology, United States) began the talk by discussing genetic-based approaches for developing new anti-malarial treatments. With the current challenge of drug resistance in malaria and relatively few compounds with novel mechanisms of action, Niles's team employed advanced technologies to manipulate gene expression in the malaria parasite and study potential target proteins, such as tRNA synthases. As a matter of fact, significant fitness defects in P. falciparum were observed when the expression of tRNA synthases was reduced. Moreover, specific chemical compounds that selectively inhibited certain members of the tRNA synthase family were identified, aiming to minimize off-target effects. In parallel, Niles's team have developed strategies to discover new molecules that act on specific targets by screening large compound libraries, which allows the identification of novel compounds interacting with validated targets and facilitates the development of antimalarial drugs. Overall, this study highlights the importance of using an integrated approach that combines genetic techniques with target-guided chemical screening to identify and validate potential drug targets for antimalarial therapies.
The conference came to an end with the conference organizers emphasizing the exceptional level of research that had been presented throughout the meeting. They also spoke on the importance of being part of a research community that shares a common goal in a collaborative nature. Furthermore, they encouraged everyone to participate in the upcoming 20th anniversary BioMalPar Conference, scheduled for 2024. The conference organizers also extended their gratitude to the EMBL and technical team for running a smooth conference.
This report is brought to you by the MESA Correspondents Faith Hungwe, Aaron Lartey and Rinter Karimi Kimathi. Senior editorial support has been facilitated by Yunuen Avalos Padilla and Alba Pérez Cantero.