New treatments for the neglected human pathogen whipworm: exploring mechanism of action at a subcellular level.

BACKGOUND: Infection with soil-transmitted helminth parasites threatens a quarter of humanity causing significant morbidity, particularly in children. The parasitic helminth Trichuris trichiura (whipworm) is no exception to this, but presents an additional challenge as current anthelmintic treatments show poor efficacy against this helminth species. No vaccine exists: new innovative drugs are urgently needed. This proposal builds on our recent identification of a novel class of drugs (the IMPs) which kill T. muris in vivo and ex vivo. However, their mechanism of action and how they are taken up into parasite tissues is unknown. Indeed, how compounds are taken up by the parasite in general is undefined, with even the feeding mechanisms and nutrient sources of the worm still debated. NanoSIMS imaging has the potential to revolutionise our understanding of the pharmacological potential of novel drug compounds by determining their subcellular localisation within the parasite and host tissue. Further, NanoSIMS also offers the opportunity to explore the basic biology of the parasite and shed light on one of the most enigmatic features of whipworm biology, the feeding mechanism.
AIMS:
1. To develop and apply NanoSIMS imaging to parasite tissues in order to localise novel drug compounds at a subcellular level.
2. To generate new knowledge surrounding the basic biology of whipworm parasites by defining the mechanism of whipworm feeding and nutrient sources
METHODS: TREATMENT OF WORMS WITH NOVEL COMPOUNDS AND LABELLED NUTRIENTS: initially we will work with the adult stage of the mouse species of whipworm T. muris. Subsequently we may include the early larval stages of the parasite and the egg stage in these analyses, the imaging of which will require additional technological development. Adult stage parasites will be grown in vivo, removed at necropsy and cultured in medium plus/minus the novel drug compound or plus/minus isotopically labelled glucose or amino acids for 24 hours. In addition, we will work with our long standing collaborators at the University of Oxford to design and synthesise derivatives of our novel anthelmintic compound series to incorporate exotic elements, or isotopic labels (e.g. 13C).

DEVELOPMENT AND APPLICATION OF NANOSIMS FOR BIOLOGICAL TISSUES: Excellent sample preparation is required for NanoSIMS analysis to achieve meaningful results as the technique operates under ultra-high vacuum. Sample preparation will be optimised by comparing chemical- and cryo-fixation to determine which preserves the in-vivo location of the drugs and structure of the cells. Samples will then be resin embedded and microtomed.

TREATMENT OF MICE WITH NOVEL COMPOUNDS OR LABELLED FOOD SOURCES: having developed the NanoSIMS methodology to detect drug compounds and labelled nutrients within individual cells of the parasite T. muris ex vivo, we will extend the work to enable a level of sensitivity such that we can detect drug compounds/labelled nutrients in worms recovered from mice fed labelled chow or dosed with the drugs in vivo. This is important as it will allow us (i) to understand actual food uptake from the host in vivo and drug accessibility and localisation within the worm in vivo, where it lives partially embedded within the host tissue. We will also be able to assess the level of drug penetrance into the host intestinal tissue which is important to inform health and safety screens and pharmacokinetic parameters