African trypanosomes sustain an infection in a mammalian host by antigenic variation, a process which involves the replacement of one variant surface glycoprotein (VSG) with a second (another) antigenically distinct VSG. Glycosylphosphatidylinositols (GPIs) are the anchors by which VSGs of varying polypeptide sequences are attached to the parasite plasma membrane. Without GPIs antigenic variation is likely to be foiled since VSG can no longer be attached to the plasma membrane. T. brucei expresses a phospholipase C (GPI-PLC) which cleaves GPIs with high efficiency. The enzyme colocalizes with GPI intermediates on the cytoplasmic side of cellular membranes, but surprisingly does not appear to cleave them. We are interested examining the regulatory mechanisms governing this apparent quiescence of GPI-PLC in vivo since the purified enzymes cleaves GPI intermediates in vitro. Our hypothesis is that constitutive activation of GPI-PLC in vivo will cause a GPI deficiency that will in turn lead to loss of cell-associated VSG. We have tested this hypothesis in Leishmania and T. cruzi and found it to be true: a GPI deficiency causes loss of the major GPI-anchored proteins gp63 and Ssp-4, respectively, in these parasites. A hopeful therapeutic approach against T. brucei which sidesteps the complication of antigenic variation involves a disruption of the mechanisms that keep the enzymatic activity of GPI-PLC quiescent in living cells. Because GPI-PLC cleaves the GPI anchor of VSG irrespective of the protein sequence (the source of the variation in antigens), the attachment of all VSGs to the plasma membrane can be prevented if GPL-PLC were constitutively activated in vivo to cleave GPI intermediates. Such a T. brucei cell line will be """"""""coat-less"""""""". If introduced into a mammalian host, the prediction is that such a cell line will be eliminated by host immune response. Any VSGs that get expressed in GPI-deficient T. brucei will most likely be secreted, and be a source of a pool of VSGs for """"""""live vaccination"""""""" if, and only if, the cell line were avirulent. In lieu of this long term aim, our specific aims are to (i) unravel the mechanisms which regulate GPI-PLC activity in vivo, and (ii) study enzymatic reaction mechanism of GPI-PLC.
| Subramanya, Sandesh; Hardin, C Frank; Steverding, Dietmar et al. (2009) Glycosylphosphatidylinositol-specific phospholipase C regulates transferrin endocytosis in the African trypanosome. Biochem J 417:685-94 |
