Trypanosome apoptotic factor mediates apoptosis in human brain vascular endothelial cells

Human African trypanosomiasis (HAT, sleeping sickness) is a devastating disease caused by Infection with Trypanosoma brucei ssp. These hemoflagellates invade the central nervous system (CNS) and induce meningo-encephalitis, neuronal demyelination, blood-brain-barrier (BBB) dysfunction, peri-vascular infiltration, astrocytosis and Apoptosis. The molecular basis of these manifestations is unclear. We previously reported T. brucei-induced Apoptosis in cerebella and brain-stem nuclei in mice at peak parasitemia. Here, we identify and characterize a trypanosome apoptotic factor (TAF) expressed by T. brucei that mediates Apoptosis in mouse-brain and human-brain vascular endothelial cells (HBVEC). Molecular, biochemical and apoptotic assays, coupled with surface enhanced laser desorption ionization (SELDI), and protein database analyses were utilized to show that TAF is a soluble, non-serum, parasite-derived, heat-labile protein that causes DNA laddering and Apoptosis in HBVEC. Protein-chip assay analysis of the SELDI spectrum of infected mouse serum and procyclic culture supernatants revealed a single major peak at 8652.7 Da. Further database analysis indicated that the TAF may be a procyclin or procyclin derivative. A synthetic 27 Mer peptide (ProEP2-1), corresponding to a region common to EP procyclins (EP2-1), induced Apoptosis in HBVEC and in cerebella of mice similar to that induced in T. brucei-infected mice. Western blot analysis utilizing an anti-procyclin monoclonal antibody (mAb) revealed that TAF is present in infected but not uninfected brain tissue lysates. Furthermore, this mAb blocked T. brucei- and ProEP2-1-induced Apoptosis in HBVEC in vitro. We conclude that T. brucei TAF or its derivative(s) play a major role in the Apoptosis associated with HAT pathology.