GPI-anchored Proteins and Free GPI Glycolipids of Procyclic FormTrypanosoma bruceiAre Nonessential for Growth, Are Required for Colonization of the Tsetse Fly, and Are Not the Only Components of the Surface Coat

Abstract

The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine-containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine-containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI-anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

“…The first four species are consistent with those reported previously, where the m/z values were assigned as C32:1-IPC, C32:0-IPC, C34:1-IPC and C34:0-IPC, respectively (Guther et al, 2006). TbSPT2 RNAi parasites attempt to compensate for the loss of IPCs by increasing the synthesis of several PC species as well as the previously identified 1-O-C18:0-alkyl-2-O-18:2-acyl-PI (Guther et al, 2006) that are not abundant in the parental cells. Further, an increase in the levels of cholesterol and cholesteryl esters upon TbSPT2 RNAi induction and myriocin treatment, presumably by increased cholesterol uptake, suggests another mechanism of compensation for bulk loss of sphingolipids.…”

“…Nevertheless, because we could not assign the specific sphingoid bases and fatty acids in any of the IPC species, further biochemical characterization of the ceramide moiety of these ion species remains to be carried out. It is noteworthy that IPC species at m/z 750, 752, 778 and 780 were also recently identified in T. brucei, and assigned as C32:1-IPC, C32:0-IPC, C34:1-IPC and C34:0-IPC, respectively (Guther et al, 2006). Moreover, the IPC species corresponding to C16:0/d18:1-IPC and C16:0/d18:0-IPC were also found to be major free IPC species in T. cruzi (Bertello et al, 1995) …”

Characterization of inositol phosphorylceramides from Leishmania major by tandem mass spectrometry with electrospray ionization

“…However, the product-ion spectrum of the ion at m/z 544 ( Figure 1a, suggesting that the MS 2 spectra arising from these scans may be applicable for profiling the IPCs in mixtures, similar to that previously described for phosphatidylinositol [30]. The profiles of the MS 2 spectra acquired by neutral losses of 260 (loss of C 6 …”

“…and Trypanosoma spp. is unglycosylated IPC [5][6][7], which accounts for 5% to 10% of total cellular lipids in Leishmania [5] and are enriched in raft-associated membrane fractions [8].…”

“…and Trypanosoma spp.) have been previously analyzed by ESI tandem mass spectrometry in the negative-ion mode [6,7,19]. Here we report more detailed structural studies towards the identification of the long-chain base and the fatty acid substituent of IPC using tandem mass spectrometry.…”