Trypanosoma Cruzi Phosphoinositide -Specific Phospholipase C (tcpi-Plc) and Its Role in Differentiation

173 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2004.A newly discovered Trypanosoma cruzi phosphoinositide phospholipase C (TcPI-PLC) is lipid modified in its N-terminus, and located in the plasma membrane of amastigotes. The N-terminal 20 amino acids of TcPI-PLC contain primary structural information sufficient to target the heterologous Aequorea victoria green fluorescent protein (GFP) to the outer surface of the parasites. Through the study of trypanosome transfectans expressing various TcPI-PLC deletion mutants fused to GFP, we found that myristoylation of a glycine residue in the second position, and acyl modification of cysteines in the fourth, eighth, and fifteenth positions of the coding sequence, are required for correct plasma membrane localization. TcPI-PLC is translocated onto the extracellular phase of the plasma membrane as indicated by immunofluorescence analysis and assays measuring the sensitivity of GFP fusion proteins to extracellular pH in transfected cells. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell. TcPI-PLC is believed to play a role in differentiation of the parasite since its expression increases during the differentiation of trypomastigote to amastigote stages. To determine if TcPI-PLC is involved in this differentiation step, antisense inhibition using phosphorothioate-modified oligonucleotides, and overexpression of the gene were performed. Antisense oligonucleotide-treated parasites showed a reduced rate of differentiation in comparison to controls, as well as accumulation of intermediate forms. Overexpression of TcPI-PLC led to a faster differentiation rate. In contrast, overexpression of a mutant TcPI-PLC that lacked the lipid modification at its N-terminus did not affect the differentiation rate. Therefore, when TcPI-PLC is expressed and targeted to the plasma membrane, it is involved with the differentiation of trypomastigotes to amastigotes, which is an essential step for the intracellular replication of these parasites.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD