Dissection of the Leishmania mannogen biosynthetic pathway: beta 1-2 mannan in pathogens and beyond

Most living organisms store energy reserves in the form of glucose-based polysaccharides; glycogen in humans, and starch in plants, for example. But some organisms use more unusual forms of carbohydrate storage. As part of an international team, we have just discovered the Leishmanial parasite - the causative agent of the sandfly-transmitted Leishmaniases, for example, stores energy in the form of an unusual polysaccharide termed 'mannogen'. Formation of this energy store is essential for the parasite infectivity. This grant will investigate how mannogen is made and broken down by Leishmania. We will dissect exactly how enzymes, nature's catalysts, are used to make this energy store and see if the enzymes can be inhibited in order to provide the academic foundation for potential new anti-parasite agents. Around two million people each year contract this debilitating disease and around seventy thousand will die. Our research will provide insights into the metabolism into this major world parasite. Beyond this, our work shows that similar enzymes exist across bacteria, our work will probe their structure and function and hopefully identify new biochemical pathways in a range of bacteria.

Eukaryotes typically synthesize one or more intracellular carbohydrate reserves that have important roles in regulating central carbon and energy metabolism, replication and growth. Typically, these storage polysaccharides are polymers of glucose; starch, glycogen, trehalose etc. We have recently discovered that Leishmania, and other trypanosomatid parasites, have evolved the ability to make a storage polysaccharide of beta-1,2 linked mannose, termed mannogen. A seven gene cluster for the enzymes involved in mannogen biosynthesis has been identified and its enzymes cloned. Genetic disruption of the mannogen cycle leads to loss infectivity of the pathogen in the mammalian host. In preliminary work, we have shown that these enzymes are a novel, new family, of carbohydrate-active enzymes with elements of glycosyltransferase and glycan phosphorylase. Work in this proposal will focus on linking the molecular aspects of these enzymes, to virulence. Work funded by the grant will provide the structural, kinetic and mechanistic dissection of this seven gene cluster for mannogen biosynthesis. We will establish robust kinetic assays, translatable to high-throughput, to provide the fundamental academic basis for enzyme activity and inhibition. We will dissect the structural basis for the essentially unique ability of different closely-related members of this family to use GDP-Mannose or Mannose-1-P donors; occasionally both. We will build on this knowledge to study the roles of similar enzymes in diverse prokaryotes, where we have recently identified these enzymes but where no equivalent beta-1,2 linked mannose is described and in pathogenic fungi such as Candida where-distantly related enzymes may be implicated in cell wall mannans. Together, the molecular work proposed will underpin study of Leishmanial virulence, link that to new pathways in bacteria and fungi, and provide fundamental insight into a unique new class of carbohydrate-active enzyme.

Summary of goals and impact
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The main aim of this research is to develop the fundamental molecular and chemical understanding of a new pathway in Leishmania, that is responsible for the synthesis of mannogen. The goal of the work is academic, but by achieving our goals we should provide a molecular platform to allow subsequent exploitation of this pathway (already genetically validated) by small molecules. We envisage enabling Leishmanial drug development based on understanding of a new pathway related to virulence, with spin-offs for anti-fungal and bacterial strategies.

To achieve this, the following impact objectives will be met:

A kinetic assay that allows us, ultimately, to approach drug discovery platforms

Three-dimensional insight into ligand binding and catalysis, such that one could ultimately consider rational drug design or lead optimisation (in future work)

Sufficient understanding of related bacterial and fungal enzymes, such that one could also begin to consider those as potential drug targets

Who might benefit and how might they benefit from this research?
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Who: Patients with leishmaniasis, their families communities and carers:

How: Ultimately, the main aim of the research is to improve patient health, by developing better ways to treat leishmaniasis. That is along way down the timeline, but beyond the academic beneficiaries, this is ultimately the goal of the work. Drugs for leishmaniasis have serious side effects, may be painful on administration and often require long treatment courses. Furthermore, the direct (drugs charges) and indirect (e.g. loss of earning to support sick family members in hospital) costs of treatment have considerable impact on populations with household incomes of often <$5 per day. Thus, research that seeks to discover new drug targets may, in the long term, lead to better treatments.

Who: Women The impact of leishmaniasis is most profound on young girls and women, particularly in regions where clothing limits sand-fly exposure to the facial regions. This may affect interpersonal relationships, marriage prospects, social activities and capacity for work. This may lead to isolation, late presentation for treatment and consequently less favourable outcomes (Al-Kamel, M.A. Int. J. Women's Dermatology. 2016. 2:93-101).

How: Better drugs would seek to reduce this societal burden

Who: Drug development agencies and funders

How: A greater understanding of the molecular basis of pathogenesis and new targets for treatment will directly benefit those involved in the development and clinical testing of new drugs such as the drugs for neglected Disease initiative https://www.dndi.org/

Who: Drug Discovery Platforms/ compound libraries

How: Compound libraries and drug screening facilities need the combination of (a) validated targets (b) robust coloured assays that allow up-scaling to medium/high throughput (c) a structural template on which to improve potential lead compounds. The goal of this work is to provide that academic framework so that such work could ultimately be undertaken.

Who: Workers on other NTDs, and on bacterial and fungal disease

How: we envisage that many of the research questions that we are addressing equally apply to other NTDs and see this project as a way to potentially re-set the translational research agenda for diseases beyond leishmaniasis. Equally, the links we seek to study to bacteria and even fungal mannan biosynthesis may open-up a new campaign for therapeutic agents in those directions.