A Drosophila-Herpetomonas model as a blueprint to study Leishmania infection in phlebotomine sand flies.

Neglected tropical diseases (NTDs) like sleeping sickness, leishmaniasis, hookworm infections, river blindness and elephantiasis are the most common infections of the world's poorest people and the leading causes of chronic disability and poverty in low- and middle-income countries. NTDs especially affect children and young women of reproductive age and consequently deprive them of their health and economic potential. NTDs also impair agricultural productivity and are an important reason why the world's poorest 1.4 billion people who live below the poverty line cannot escape destitution and despair. For example, human sleeping sickness caused by african trypanosomes is endemic to 36 countries in sub-Saharan Africa with 70 million of the 400 million inhabitants at risk. The continent is recovering from an epidemic (300-500 000 cases in 1998) and recent estimates suggest that there are currently 50-70 000 people infected per year [World Health Organization (WHO) report, 2006]. Sleeping sickness is fatal if left untreated. The related disease (nagana) in domesticated animals causes estimated losses to African agriculture of US$4.5 bn per year and has had a profound effect on development of the continent. However, this is not a problem in one continent: Leishmaniasis, caused by several species of Leishmania is endemic in India, Bangladesh, Nepal, Sudan and South and Latin America, while the existence of an underbelly of Leishmaniasis among the poorest people in the South of the USA came recently into sharp focus.

Despite the devastating effect of these diseases on health and development, with evidence that their global burden is as great as that of any other serious disease, financial support for control and elimination efforts, as well as research and development (R&D), have been inadequate. Indeed, in Millennium Development Goal 6 (to "combat HIV/AIDS, malaria and other diseases"), NTDs were not even specifically mentioned but merely considered as part of the "other diseases" (United Nations Report 2009). However, policy makers are slowly beginning to appreciate the importance of NTDs. WHO has a new Department of Neglected Tropical Diseases (WHOTDR), which has a new 10-year strategic plan with support from UN agencies, member states, and private philanthropies. However, an integrated offensive to tackle NTDs is needed. Action must be taken both at the field and drug delivery level as well as at the R&D level. There are a lot of things we are unable to understand about NTDs during the transmission process in the invertebrate host and the life of the parasite inside its insect vector, however working with the insect vectors themselves has proven to be very difficult in technical terms (developing of colonies in the lab, genetic transformation, functional genetics and genomics).

This proposal suggests going back to the basic biology of the insect-parasite interaction by using a model "hopping" approach. The fruit fly Drosophila melanogaster has proven informative in immunity studies with its relevance and contribution recognized in the 2011 Nobel prize for Physiology and Medicine. However, the model "hopping" approach that we propose here is a comparative approach, which goes far beyond the level of generic innate immune signalling and looks at comparing gut-specific responses of insect vectors like sand flies (Leishmania vector) following parasite infection with the more easy to culture and do experiments with, fruit fly. We will uncover by DNA microarray experiments and tissue specific RNAi, the host genes responding to Herpetomonas gut infection in fruit flies. We will then knock-down those conserved in sand flies following Leishmania infection. Drosophila and sand flies belong to the same order (they are dipteran insects) and comparing them offers the potential to uncover evolutionary conserved or contrasting functional characteristics in their immune response towards their respective protozoan parasites.

For the vast majority of vector borne parasites the ability to overcome the insect midgut defences is central to transmission. However, for many such diseases we know virtually nothing about the molecular mechanisms involved. For vectors such as tse-tse flies and sand flies the prospect for rapidly improving our understanding of key interactions occurring in the midgut when challenged by parasites, is bleak. This is because the 'tool box' required untangling the interactions is very unlikely to be rapidly developed. For example, there is no realistic prospect of producing transgenic technology for tsetse flies because eggs are inaccessible due to intrauterine development of larvae; maintenance of multiple lines of either tsetse or sand flies permitting genetic studies is impossible because of the cost and complexity of culturing colonies; bioinformatic resources are still in their infancy. In this application we suggest that under these circumstances a comparative approach in which kinetoplastid interactions in Drosophila melanogaster are studied in the first instance, will permit us to make significant progress in understanding the more important cases of insect-parasite interactions (in this case Leishmania in sandflies). Herpetomonas ampelophilae is a natural kinetoplastid parasite of D. melanogaster, which establishes infection in the midgut of the fly and can go on to invade the salivary glands. We now have this protozoan in culture and intend, through a combination of genomics, cell biology and RNAi experiments to identify the gut-specific host genomic contingent involved in parasite challenge and compare to sandflies. In addition, we will study the interaction between the indigenous flora and the parasite and the role of the former in protecting the host from parasite infection. These studies will outline the major immune pathways by which insects respond to kinetoplastid challenge in the midgut and the role of the gut flora in determining vectorial capacity

It is the first time that a model "hopping" approach will be tried in the context of Neglected Tropical Diseases (NTDs). This approach will transform the limited and fragmented knowledge we have of the genes involved in the gut response of important NTD insect vectors to parasite challenge. This transformation will stem from the fact that for the first time we are going to have a global, functional view of the gut-specific host genomic contigent involved in response to parasite challenge. Our holistic approach is also taking into account the interaction between the intestinal microflora and the parasite and how this interaction is influenced by changes in host gut-specific gene expression.
It is important to note that the data of the sand fly genome (a project in which co-applicant Dillon is the major player) are just going to become available in their annotated form giving a sense of urgency to the project. Finally, from the fruit fly host perspective, the proposed work will be the first time that a kinetoplastid parasite-Drosophila interaction will be analysed at the molecular level and the results will be a reference point for comparisons of similar host-parasite interactions in other Diptera (e.g. tsetse flies and mosquitos).