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Sophie Collier completed an honours project under the supervision of Dr. Matthew Dixon and Prof. Leann Tilley at the University of Melbourne, characterising the role of formin proteins in P. falciparum invasion and gametocytogenesis. She is currently wrapping up her PhD project in Prof. Geoffrey McFadden’s lab at the University of Melbourne. Her PhD project has focused on organellar inheritance and paternal leakage of the apicoplast and mitochondrion in P. berghei.
“Locked Out and Left Behind: A Study of Organellar Inheritance in P. berghei”
Plasmodium parasites harbour a single mitochondrion and a single relic plastid (apicoplast) at each stage of their life cycle. Both organelles are essential and used as drug targets. Previous genetic cross studies indicate that both organelles are maternally inherited during mating, but the precise mechanisms underpinning such uniparental inheritance remains unknown. To investigate organellar inheritance in a sex-specific manner, we have developed single sex P. berghei lines with fluorescently tagged apicoplasts and mitochondria. Using lattice light-sheet and expansion microscopy, we show that the mitochondrion and apicoplast are absent from newly formed male microgametes through exclusion and degradation mechanisms executed during exflagellation. In turn, live-cell microscopy reveals the presence of an elongated, perinuclear positioned apicoplast and an expanded mesh-like mitochondrial network that cradles the nucleus in activated female gametocytes. To explore whether the organellar genome is degraded prior to elimination, we used digital droplet PCR analysis to show there is a substantial decrease in the copy number of the apicoplast and mitochondrial genomes in male gametocytes compared to females. Maternal inheritance systems sometimes fail—a bit of ‘Adam’ mitochondrial DNA occasionally gets through in humans for instance. To test for paternal leakage in malaria parasites, we set up a forced cross with a selectable polymorphism in the mitochondrion of the male parent. After screening 1.9 million sporozoites across seven crosses, we identified a single male leakage event, thus demonstrating for the first time that drug resistance encoded by the mitochondrial genome of malaria parasites can, very infrequently, be inherited from the male parent. Overall, this work helps to better inform future therapeutic strategies targeting these organelles and improves our understanding of how organelle encoded resistance mutations are transmitted and how this might impact malaria treatment.
Lee Yeoh is a post-doctoral Fellow at Burnet Institute in Melbourne. Lee completed a BSc(Hons) at the University of Melbourne, majoring in Botany. He then completed a PhD in the laboratories of Associate Professor Stuart Ralph and Professor Geoff McFadden, followed by a postdoctoral position with Dr Michael Duffy, both at the University of Melbourne. His research has combined equal parts bioinformatics and wet-lab work, including transcriptomics, whole-genome sequencing, molecular and cell biology, and CRISPR-Cas9. His projects have investigated the role and mechanism of alternative splicing in apicomplexans, and the function of histone modifications and epigenetic regulation of transcription in the malaria parasite. He joined Professor James Beeson’s lab in 2021 to research the immune response to Plasmodium vivax malaria, including research into monoclonal-antibody technology.
“Monoclonal antibodies and targeted vaccine design against malaria”.
Monoclonal antibodies have recently emerged as effective therapeutics against various diseases including cancer, autoimmune diseases, and COVID-19. Vivax malaria has been understudied, and there are limited prophylactics or therapeutics available. Monoclonal antibodies have been touted as a new breed of weapon against vivax malaria.
We recently cloned and expressed over 20 monoclonal antibodies specific to a malaria invasion ligand (PvAMA1). Promisingly, a number of these strongly inhibit parasite invasion in vitro. We identified an antibody that was most potent against different strains in vitro. Further structural studies suggest that this antibody prevents a conformation change necessary for the ligand to bind to its partner during invasion.
We also tested these antibodies with assays that test downstream immune responses, which are better associated with protection compared to invasion assays in vitro. Many of the inhibitory antibodies were also capable of stimulating strong downstream immune responses, suggesting that many of our monoclonal antibodies are also effective in vivo.
Monoclonal antibodies can allow precise identification of specific epitopes of antigens. We observed high reactivity to a poorly-characterised part of our antigen. We speculate that this may demonstrate a novel ligand important in invasion. While this antigen has previously been investigated as a vaccine target, these developments imply the existence of an important novel region of an existing essential invasion apparatus; this may be an additional target for new vaccines or drugs.
We have identified promising novel monoclonal antibodies with potent activity, which can be prioritised as potential therapeutics. We are also elucidating the role of various domains of the antigen, potentially identifying additional drug targets or targets of immunity.
Our ASP Online Seminar Series image is created by Thorey Jonsdottir.
