Functional proteomic analyses of tsetse-trypanosome interactions.

African trypanosomiasis is a major public health and agricultural problem throughout much of Africa. The disease affects both man and his domestic animals causing specific disease and ultimately death in both. In humans the disease is more commonly called African sleeping sickness and if left untreated is fatal. Sleeping sickness is present in 36 countries in sub-Saharan Africa, more than half a million people currently have the disease and sixty million people are at risk of becoming infected. African sleeping sickness is caused by a protozoan parasite called a trypanosome, which is transmitted to man (or other vertebrate hosts) by the bite of a tsetse fly (Glossina spp.). Once bitten by an infected tsetse fly the disease begins to take hold. In the beginning patient's may experience few symptoms but the grip of sleeping sickness is deadly and the process of dying from this disease, horrific. The parasite enters the bloodstream of the host from the bite site via the lymphatic system. Once in the bloodstream, the parasites proliferate. The host mounts an immune response but the battle is futile. The parasite escapes from the host's immune response by changing the antigens displayed on their surface (the variant surface glycoproteins of their antigenic coat). The host fights back making new antibodies against the new antigen on the parasite but the parasite changes the antigen again - continually moving the goal posts. While the parasite is busy evading the host's immune system, the host does not escape so easily. The waves of changing parasites in the blood cause systemic fever, debilitation and wasting. Without treatment a second stage follows when the parasite crosses from the blood into the brain resulting in distressing changes in the sleep cycle, mood, personality and behaviour of the patient. Other higher mental functions are disrupted and, as the disease progresses, mutism and severe wasting ensue. Patients finally succumb to the disease often as a result of a secondary bacterial infection. Drugs are often not available, those that are, are expensive, require a prolonged treatment regime which is brutal on an already struggling body and the drugs can have serious adverse side effects. Current treatment involves the use of an arsenic based drug which in itself is responsible for the death of 4% and 12% of the seriously ill patients who receive it. No vaccines are available and it is unlikely they will be developed for trypanosomiasis because of the complex biology of the parasite. African trypanosomiasis affects humans in a second way by causing disease in domesticated animals. Human sleeping sickness is closely related to a widespread infection of cattle known as N'gana, which has seriously restricted cattle rearing in many areas of Africa. N'gana is also responsible for restricting agricultural development in tropical Africa by limiting the use of pack and draught animals.

One important way of controlling both African sleeping sickness and N'gana is by controlling the infection of the vector, the tsetse fly. In order to be able to control the infection in the vector or control the vector itself, we need to understand how the parasite interacts with the vector. Our investigations are helping us to improving the understanding of these vector and parasite interactions. However, there have been many obstacles in the way. Very little molecular information was known about tsetse flies until our collaborators, the Lehane laboratory in Bangor undertook a gene discovery project with the Sanger Centre in Cambridge.  They produced 21, 427 expressed sequence tags (ESTs) from G. m. morsitans adult midgut and have assigned putative functions to a significant proportion of them by homology searching. 68 genes with putative immunity related functions were selected and macroarrayed and their expression profile determined following bacterial or trypanosome challenge (read PDF of complete article here).

Whilst the EST project described above has made a major contribution to our understanding of tsetse biology and tsetse-trypanosome interactions, the macroarray studies were performed on mRNA of the putative immunity genes. Consequently we have no direct evidence that the proteins and peptides encoded by these genes and others are, in fact, found in the gut lumen and therefore available to interact with infecting trypanosomes. There is increasing evidence that many genes in the midgut of blood-sucking insects are post-transcriptionally regulated and so meaningful data on the gene product levels can only come from analyses of the proteins and peptides themselves and this is what we are now doing.

We are aiming to identify a panel of immune related proteins and subsequently their genes in the important trypanosome vector Glossina morsitans morsitans. This is an integrative functional proteomic approach to understand the mechanisms of immune function and disease pathogenesis in the tsetse fly, combining novel proteomics technology (in particular Difference Gel Electrophoresis, DIGE) with molecular biology and immunology. We are using a proteomics approach to identify immune related proteins and cDNA library screening to identify the genes encoding the proteins of interest. Our investigations will determine the changes in the expression levels or post-translational modifications of proteins in response to trypanosome challenge. This information will provide valuable insights into the factors determining the outcome of trypanosome infection in tsetse flies and determine the changes in the expression levels of these immune related proteins or post-translational modifications of them in response to trypanosome challenge. 

                                                                                                                                                                            2D DIGE gel

This project has strong collaborative links with Professor Mike Lehane (Bangor, UK), Professor Wendy Gibson (Bristol, UK) and Dr. Serap Aksoy (Yale University, USA).