Study On Refractoriness Of Tsetse Flies Carrying Recombinant Symbionts Expressing An Anti-trypanosomal Product

Tsetse flies transmit human sleeping sickness (Africa trypanosomiasis) in tropical Africa. It is also the vector that causes Nagana in livestock. This is a great burden for health and development in Africa. It is impossible to develop -a specific antibody because of antigenic variation on the surface of trypanosomes. To date, the most effective approach to control the disease is vector control (tsetse fly) .Immune response research shows one of the antimicrobial peptide, Attacin, was upregulated expressed in the tsetse fat body after trypanosome infection. But the infection was not effectively cleared due to the late response or expression site discrepancy (fat body but not midgut) . The infection rate was significantly decreased if we upregulate Attacin expression by E.coli injection prior to trypanosome feeding.Attacin gene was cloned and expressed in Drosophilla S2 cells. Purified protein shows strong activity against E.coli, as well as trypanosome. But tsetse symbiont Sodalis, a gram negative bacterium, was quite resistant to Attacin. Attacin affected both procyclic and bloodstream trypanosome. Meanwhile, feeding tsetse with Attacin shows that there is no fitness lost, but attacin-feeding tsetse reveals significantly low trypanosome prevalence in midgut.Because of its unique viviparous reproduction system, we cannot obtain the fertilized egg to do transgenic experiments. Since Sodalis is quite resistant to Attacin, we tried to express Attacin in Sodalis. Result shows that intracellularly accumulated peptide causes cell lysis and growth retarded. But released protein has a killing effect on trypanosome both in vivo and in vitro.Thus, we construct a recombinant plasmid that has Sodalis endogenous promoter and signal sequence to express Attacin in Sodalis. Extracellular secretion of peptide mediated by signal peptide could improve cell growth. Microinjection of recombinant Sodalis that Attacin was expressed into tsetse shows their offspring has significant lower midgut and, more important, salivary gland infection rate.To obtain genetic stable strain, we evaluated the clearance of symbionts in tsetse, impact of vitamin supplement on fly fertility and relationship between symbiont and trypanosome infection.