title: The influence of tsetse midgut oxidants on trypanosome establishment
Creators: name: Ramphul, Urvashi
Publisher: name: University of Liverpool Repository
Year: 2012

description: Tsetse flies (Diptera: Glossinidae) are the vectors of African trypanosomes which cause human sleeping sickness and nagana to animals. Despite their importance as vectors, tsetse flies are largely refractory to trypanosome infections after the first two bloodmeals. Fly immunity is thought to play a crucial role in determining the outcome of initial parasite establishment in the fly midgut. As a result of defending against trypanosomes and haem released during blood digestion, redox genes probably play a significant regulatory role in midgut physiology of tsetse flies. The primary objective of this PhD thesis was to test the hypothesis that superoxides are a core protective device for tsetse flies against invading trypanosomes. To first examine whether inducible nitric oxide synthase (NOS), a putative pro-oxidant enzyme, acts as one of the barriers to trypanosome establishment in the tsetse midgut, temporary depletion of NOS from the midgut using RNA interference (RNAi) was performed. This caused a significant increase (p=0.011) in midgut infection prevalence (2-fold) compared to when NOS was reduced post-infection. It had the most noticeable effect when knockdown occurred before the trypanosome infection event. Further evidence for this was given by the inhibition of NOS by oral administration of the NOS inhibitor L-NAME which also caused a significant increase in midgut infections (p=0.048). Further, indirect evidence of the anti-trypanosomal role of NOS is seen in the fact that NOS activity increased almost 2-fold in trypanosome-infected flies compared to uninfected flies. By contrast, in tsetse NOS levels did not change in response to bacterial infections with Gram-negative Escherichia coli or Gram-positive Staphylococcus aureus suggesting this is quite a specific response to trypanosome infection. An indirect approach was taken to measure reactive oxygen species (ROS) levels in tsetse flies. ROS levels were correlated with trypanosome prevalence in infected flies under various physiological conditions known to impact susceptibility: fly age, starvation, fly sex, bloodmeal fractions, immune-stimulation with bacteria, knockdown of immune genes (tsetseEP protein) and puparial conditions. The indirect evidence gathered suggested that changes in ROS levels cannot explain changes in susceptibility seen in tsetse under this range of physiological conditions. The only result suggesting a role for ROS in trypanosome infection was the observation of a significant (p=0.029) increase in hydrogen peroxide levels in flies infected with live trypanosomes compared to uninfected flies. Consequently, a direct approach was attempted to look for a role of ROS in trypanosome establishment in tsetse midgut. Gene knockdown via RNAi of genes involved in ROS generation (dual oxidase – Duox) and removal (oxidation resistance 1 - OXR1) was attempted. Knockdown of transcript levels of Duox resulted in increased midgut infection prevalence suggesting its involvement in controlling parasite establishment. However, despite repeated attempts, transcript levels of OXR1 could not be knocked down. A bioinformatic approach including phylogenetic analyses of NOS, Duox and OXR1 was taken for comparative analysis with other dipterans. In addition, manual extension of the sequences of these genes was carried out and the extended protein sequences of these genes are now comparable in length to Drosophila orthologs, incorporating necessary domains and residues essential for their functionality. Available resources such as a 454 cDNA tsetse midgut transcriptome enabled the identification of 70 genes which were differentially expressed in tsetse fed trypanosomes. Trypanosome infection was found to have a substantial effect on metabolic and cellular processes in the fly, in addition to the emerging common classes such as serine proteases, adhesion proteins, reactive intermediates and immune-related genes. Finally, with the recent release of the Glossina morsitans morsitans genome, a selection of antioxidant genes was manually annotated to include transcript and protein sequence predictions. Consequently, a comparative analysis with the model system, D. melanogaster was carried out. The essential components of the antioxidant system appear to be conserved, with a few exceptions. In addition, lack of differential expansion of antioxidant gene families among Glossina and other dipterans including Drosophila reflects their close relation. Furthermore, we are now able to validate antioxidant genes identified in the past and find novel genes such as a second thioredoxin reductase in tsetse. Overall, this research involves both functional and bioinformatic approaches to offer new insights into the roles of oxidants in response to trypanosomes in the tsetse fly.