Expression and functional analysis of lipids and glycolipids from the mammal-dwelling stages of Trypanosoma cruzi

Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions. Glycosylphosphatidylinositol (GPI)-anchoring is a protein post-translational modification ubiquitously found in eukaryotes. There is a growing body of evidence showing that protein-free GPIs (or glycoinositolphospholipids, GIPLs) and GPI-anchored proteins (GPI-APs) are involved in host-protozoan interaction processes, such as host-cell adhesion and invasion, and pathogenesis. Here, we used a highly sensitive and unbiased approach that employs liquid chromatography-tandem mass spectrometry (LC-MSn) for the analysis of the GPIome (GPIomics) of the mammal-dwelling trypomastigote and amastigote stages of Trypanosoma cruzi, the causative agent of Chagas disease. This approach allows for the structural characterization of both the lipid and the glycan moieties of GPI-APs and GIPLs. We have identified over 140 GIPL and GPI-AP species from these two parasite forms, most of which had not been described in the literature. In contrast to epimastigote-derived GIPLs (eGIPLs), trypomastigote-derived and amastigote-derived GIPLs (tGIPLs and aGIPLs, respectively) tend to have longer and structurally more diverse glycan moieties. Similar results were observed for trypomastigote-derived and amastigote-derived GPI-APs (tGPI-APs and aGPI-APs, respectively), although tGPI-APs and aGPI-APs tended to be structurally less diverse than their GIPL counterparts. The lipid moieties of tGIPLs are composed mainly of O-alkyl-2-O-acyl-glycerolipids (AAGs), typically with longer fatty acid chains than those of eGIPLs. Conversely, amastigotes tend to have an approximately equal number of GIPLs containing AAG or ceramide moieties. Interestingly, the majority of the lipid moieties of GPI-APs derived from both mammal-dwelling stages of T. cruzi contain C18:1- or C18:2- fatty acid substituents. The proteomic analysis of fractions enriched in GPI-APs from the three life-stages of this parasite showed much greater protein diversity in the mammal-dwelling stages than in epimastigotes. The observations made in this study will, hopefully, help us further understand possible structure-function correlations of GPIs and their role during chronic infection