Production of infective Prypanosoma brucei brucei expressing green fluorescent protein for localisation of trypanosomes in brain of infected rodents

Abstract

Human African trypanosomosis (HAT) is caused by subspecies of T. brucei found in East, Central and West Africa, with T. b. rhodesiense and T. b .gambiense causing sleeping sickness in human through the bite of a tsetse fly of the genera Glossina. The treatment of CNS infection is difficult and at the same time the biology of late stage trypanosome infection is poorly understood. In addition, the parasites hideout in the brain and their mode of survival and multiplication in the brain remains elusive. The current study aimed at production of infective T. b. brucei expressing green fluorescent protein to track the trypanosomes in infected rodents to provide a better understanding of how the parasite establishes infection of the CNS and thus causes pathogenesis within the brain. Results from the study indicate that it was possible to extract the eGFP gene, cloning it to pJET1.2 subcloning vector and thereafter into pLEW100 an expression vector before transfecting the gene into the trypanosome genome. This resulted in the production of infective trypanosome parasite expressing green fluorescent protein that can be used to track the trypanosomes in brains of trypanosome infected rodents during various stages of infection in order to provide a better understanding of how the parasite establishes infection in the CNS and thus causes pathogenesis within the brain. The tool can thus be used to connect CNS infection and scientific evidence that can be used to devise new strategies for the management of late stage African sleeping sickness