Tsetse flies (Glossina spp.) harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse’s intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. Previous experiments indicate that female tsetse flies can be successfully colonized with recombinant Sodalis, and that the paratransgenic host flies exhibit no subsequent decrease in fitness. The current study was performed to obtain a better understanding of the dynamics of recombinant bacteria establishment in tsetse. To this end, we septically infected flies with Sodalis and a non-pathogenic strain of E. coli (K12) that were genetically transformed to express the luciferase marker gene. While the Sodalis were able to establish stable infections in tsetse, the E. coli had an overwhelmingly toxic effect that resulted in host death. In contrast, similar experiments performed with Drosophila show that this insect is able to effectively clear an E. coli infection. In light of the unique effect E. coli infection has on tsetse, a functional system was developed to test our hypothesis that structural disparities within Sodalis and E. coli outer membrane protein A may be partly responsible for this fly’s unique reaction to infection with these bacteria.