Functional characterization of Glossina morsitans morsitans saliva in the context of African trypanosome transmission

Tsetse flies are important disease vectors, responsible for the transmission of African trypanosomes causing human sleeping sickness and veterinary infections in the African continent. During the obligate blood feeding interaction, tsetse flies deposit a complex salivary mixture that might inhibit host defences and support efficient trypanosome transmission. As such, this PhD primarily assessed immunological and molecular aspects of the tsetse fly/host interaction. Immunological analyses demonstrated that the major 43-45 kDa protein fraction (tsetse salivary gland protein 1 and 2- Tsal1 and Tsal2) of Glossina morsitans morsitans saliva induces strong humoral responses in mice and humans. using prokaryotic expressed Tsal1 and Tsal2, an immunological assay for human tsetse fly exposure was tested and found applicable for eventual epidemiological surveys. Additional analysis revealed that saliva exposure in mice results in a Th2 biased cytokine profile ad associated IgG1 and IgE antibody production. These antibody responses did not affect the fly's blood feeding efficiency nor its survival, suggesting that tsetse flies might prevent the outcome of detrimental anti-vector immune responses. Supporting this hypothesis, reduced murine T and B cell responses were recorded in co-immunization experiments against heterologous antigens. Infection experiments additionally demonstrated that tsetse fly saliva accelerates a Trypanosoma brucei infection onset, associated with reduced local inflammation and diminished systemic production of the trypanocidal cytokine TNF. Surprisingly, a history of repeated tsetse fly exposure also enhances trypanosome expansion in the early infection phase. To unravel the tsetse salivary composition, a G.m. morsitants lgt11 salivary gland expression library was generated and subjected to immune screening. Using this approach, full length cDNA clones encoding three unknown secreted salivary components were identified. In addition, detailed in silico analysis revealed significant homology for Tsal1 and Tsal2 with the CD39 "apyrase conserved region1" (ACR1) and the shrimp Marsupenaeus japonicus nuclease. Eukaryotic protein expression, activity analysis and in vivo RNA interference, functionally characterized Tsal1 and Tsal2 as representatives of a novel class of salivary enzymes in blood feeding insects with combined apyrase and nuclease activities. Collectively, this research offered new insights in the tsetse/host-interaction and trypanosome transmission.