Conference Agenda

Oxythiamine, an antiplasmodial thiamine analogue, kills P. falciparum via a mechanism that likely involves its conversion by thiamine pyrophosphokinase (TPK) into the antimetabolite oxythiamine pyrophosphate (OxPP). To deepen our understanding, we used in vitro evolution to generate oxythiamine-resistant parasites. Whole-genome sequencing identified a point mutation in PfTPK at position 284. Homology modelling suggested that the mutation may decrease ATP/AMP binding and consequently reduce PfTPK activity. Consistent with this, oxythiamine-resistant parasites accumulated five times less [³H]thiamine/[³H]TPP than wild-type parasites, and the activity of oxythiamine against parasites expressing a GFP-tagged version of mutated or wild-type PfTPK was altered. To investigate the role of TPK throughout the parasite's life cycle, we generated P. berghei parasites lacking TPK (PbTPK-KO). No defect was observed in PbTPK-KO during their blood stage, but they were resistant (5-fold) to oxythiamine in vivo. Although PbTPK-KO produce a similar number of oocysts compared to wild-type parasites, PbTPK-KO oocyst failed to mature and did not produce sporozoites. Transmission electron microscopy (TEM) of mosquito midguts infected with PbTPK-KO parasites revealed small oocysts containing large empty spaces and clustered membrane whorls. In conclusion, we reveal a crucial role for TPK in the antiplasmodial activity of oxythiamine and the development of P. berghei oocysts.

Pantothenate, a precursor of the enzyme cofactor coenzyme A (CoA), is an indispensable nutrient for Plasmodium falciparum. Pantothenate kinase (PanK) catalyses the first step of CoA biosynthesis. P. falciparum expresses two PanKs, PfPanK1 and PfPanK2. To date, all PanKs with a solved structure function as homodimers. In contrast, we have shown that P. falciparum and T. gondii possess a novel heteromeric PanK complex. We performed site-directed mutagenesis of key residues in PfPanK1 and PfPanK2 predicted to be involved in active site stabilisation. Parasites expressing each mutant protein were generated, from which the complex was then purified and analysed. Heterologous expression of the PfPanK complex was attempted by using the insect cell protein expression system, with all components of the complex being expressed within the same cells. Although some of the expressed components aggregated, enough remained soluble, naturally formed the complex in situ and, crucially, when purified, the complex was functional. In addition, we generated PfPanK1 or PfPanK2 inducible knockdown parasite lines and show that knockdown of either PfPanK1 or PfPanK2 is detrimental to parasite proliferation. This research advances our knowledge of a novel P. falciparum protein complex and may facilitate the identification of new drugs targeting P. falciparum pantothenate utilisation.

Ascaris infection, a prevalent neglected tropical disease, affects 819 million people worldwide and causes significant economic losses in the livestock industry. Despite available anthelmintics, reinfection and emerging drug resistance are major concerns. Targeting the early stages of infection, specifically hepatopulmonary migration (HPM), where larvae migrate from the intestine to the liver and lungs, probably guided by chemosensation, presents a promising intervention strategy. However, our understanding of chemosensation in parasitic nematodes is limited. We explored the role of chemosensation in guiding larval Ascaris suum during infection. We conducted tissue-specific transcriptional studies, identifying a chemosensory pathway specifically present in the head and amphidal tissues of Ascaris. Subsequent larval migration assays demonstrated the chemotactic responses to pig liver and lung homogenates, as well as extracted metabolites. Transcriptional profiling of the stimulated larvae highlighted the molecular pathways involved in Ascaris chemotaxis. Additionally, we developed and characterized a polydimethylsiloxane (PDMS) microfluidic device to further investigate the chemosensory behaviour of Ascaris larvae. Our findings in microfluidics revealed distinct behavioural responses of larvae to linear concentration gradients and confirmed chemoattraction to pig liver homogenates, demonstrated by increased forward speed and reduced turning. Future studies will aim to identify target-receptor interactions to potentially block chemotaxis and disrupt HPM.

Inflammatory bowel diseases (IBD), encompassing ulcerative colitis and Crohn’s disease, are chronic inflammatory conditions affecting millions globally, with no current cure. Pediatric IBD often manifests more aggressively than adult-onset IBD, accounting for 25% of total cases. These heterogeneous diseases are prevalent in Western countries, where high levels of sanitation have reduced exposure to organisms that influence immune development and response, such as gastrointestinal hookworms. These helminths secrete diverse bioactive molecules with immunomodulatory and anti-inflammatory properties. We expressed individual hookworm-derived proteins in our lab to evaluate their therapeutic potential in inflammatory conditions. One recombinant protein significantly reduced disease severity and markers of inflammation in a mouse model of experimental colitis induced before sexual maturity. Our current research focuses on elucidating the mechanism of action of this protein by identifying its target cells, assessing its impact on intestinal barrier integrity, and examining its influence on intestinal immune processes.

Leishmaniases are caused by at least 21 species of Leishmania and significantly affect human and animal health in tropical and subtropical regions. Understanding their complex epidemiology has been hindered by traditional techniques’ inability to capture the full diversity of Leishmania species. We developed a metabarcoding tool targeting the heat shock protein 70 gene using a portable Oxford Nanopore Technologies (ONT) MinION device to establish the diversity of Leishmania species infecting vertebrate hosts and vectors. We also created a new database and used it alongside the NanoCLUST analysis pipeline. This tool allowed the accurate classification of all 14 Leishmania species tested. Using this method, all Leishmania species were separated from each other, including those within the same complex, with percentage identity scores between 99.99 - 100% when compared to reference sequences. The species tested represented four Leishmania subgenera: L. tropica, L. aethiopica, L. major, L. gerbilli, L. infantum, L. mexicana, and L. amazonensis (subgenus Leishmania); L. guyanensis and L. panamensis (subgenus Viannia); L. orientalis (subgenus Mundinia); L. tarentolae (subgenus Sauroleishmania). This nanopore sequencing method provides a useful tool for the differentiation of Leishmania species to support epidemiological studies and the prevention and control of this disease in humans and animals.