Detection and characterization of novel proteins in Trypanosoma cruzi

Sammanfattning: Trypanosoma cruzi is a flagellated protozoan parasite. It infects a wide range of mammals, including humans. Human T. cruzi infections are endemic to South and Central America. The parasite is transmitted to humans mainly through an insect from the Triatominae subfamily, which feeds from mammals and defecates at the site of the wound, which allows the parasites in the faeces to infect the damaged cells at the bite area. The disease is named Chagas disease in honour of Carlos Chagas who first detected the parasites in the insect, and in this way determined that the vector of the disease was the triatomine bug. There is no cure for Chagas disease and if it is left untreated it can be lethal in the initial stage of the infection, especially for children. If the affected patients develop the chronic form of the disease, there is a high risk of organ deterioration due to the long term presence of the parasites. Only two drugs are used at present to treat Chagas disease, Nifurtimox and Benznidazole. These drugs were developed in 1960s-70s and they can cause severe side effects. Although preventive and control measures have been effective to reduce transmission, there are still 15,000 deaths per year and over 20 million people are at risk of contracting the disease. Chagas disease is one of the socalled neglected tropical diseases, for which there is little interest from pharmaceutical companies to develop new drugs. In response to the critical need for new safe and effective drugs, much research has been performed by many groups in the field, in order to expand the knowledge on T. cruzi biology and to study different enzymes and metabolic pathways that differ from humans. My aim in this thesis was to detect novel proteins in T. cruzi and characterize them by means of: assessing their localization, determining their enzymatic activity, infering putative identity, if unknown by homology comparisons, and determine if they were really expressed in the parasite. In paper I, we detected a polyprenyl synthase in the T. cruzi EST database. We have expressed and characterized this protein. The enzymes of the polyprenyl synthase family are involved in the synthesis of isoprenoids, which are essential for cell function. The identified protein was a solanesyl diphosphate synthase (TcSPPS) that had all the conserved motifs of the family, presented polyprenyl synthase activity and synthesized the maximum chain isoprenoid, solanesyl diphosphate. Long chain isoprenoids are used in ubiquinone biosynthesis. This was shown by the ability of TcSPPS to complement an E. coli strain deficient for ubiquinone production. By using immunofluorescence microscopy, immunogold electron microscopy and cell fractionation we localized TcSPPS to the glycosomes, a peroxisome-like organelle of T. cruzi. In paper II, we report the localization of a short polyprenyl synthase, farnesyl diphosphate synthase (FPPS) in both T. cruzi and Trypanosoma brucei, a closely related trypanosome to T. cruzi that causes sleeping sickness in Africa. Short chain polyprenyl synthases produce short isoprenoids that are utilized to for example modify signalling proteins that utilize the isoprenoid arm to attach to membranes and receptors. As we found the TcSPPS in the glycosomes, we wanted to determine if the entire part of the isoprenoid pathway where these two enzymes take part, was compartmentalized to the glycosomes or not. We found that this was not the case. Both TcFPPS and TbFPPS are present in the cytoplasm. In paper III, we partially characterized a third polyprenyl synthase of T. cruzi, TcPPS. We detected this protein by western blots analysis in the three stages of the parasite and in the cytoplasm of epimastigotes in T. cruzi. It is an unusual protein, due to the 747 amino acid sequence and a molecular weight of 85 kDa, compared to the usual size for the family, which is around 40 kDa. Another particular feature was the presence of a domain of unknown function, DUF2006, which is unrelated to the conserved polyprenyl synthase conserved motifs. We hypothesized that this enzyme could be a GGPPS. The recombinant TcPPS had polyprenyl activity, but the preferred substrate was GPP instead of FPP, the preferred substrates of GGPPSs. In paper IV, we used a mass spectrometry based proteomic approach to detect novel proteins in an organelle enriched sample from T. cruzi epimastigotes. The organellar proteins were separated by 2DGE and 1DGE and subsequently subjected to LC-MS/MS. The results from mass spectrometry were used to search against the T. cruzi translated genome. The search rendered 396 protein identifications. For 173 of them, this was the first expression data reported in T. cruzi. Furthermore, the proteins in the sample belonged to several organellar compartments and the level of cytoplasmic and highly abundant surface proteins was much reduced. We located five novel proteins to the acidocalcisome, mitochondrion, ER and cytoplasmic vesicles, through immunofluorescence microscopy of epitopetagged over-expressed clones. In summary, this work has contributed to the detection and characterization of several novel proteins in T. cruzi, and has answered various questions and has generated new hypotheses to be tested.