Authors
A M Murillo2; S Marsiccobetre2; K V Sowerby1; E Pohl1; A M Silber2; 1 Durham University, UK; 2 Biomedical Science Institute - USP, Brazil Discussion
Trypanosoma cruzi can use amino acids as energy sources and to support several biological processes such as differentiation, resistance to stress conditions and host-cell invasion. Metabolites containing -SH groups (such glutathione, trypanothione, cysteine, and some of its intermediates such as cystathionine) are relevant to buffer the redox state of the different sub-cellular compartments of this organism. The cysteine synthase (CS) catalyze the second step in the de novo biosynthesis of cysteine, however, its role in redox homeostasis has been unexplored in T. cruzi. In this work, we identified a T. cruzi sequence encoding a functional CS (TcCS), which was cloned, expressed and affinity purified. We are currently pursuing the 3D structure by X-ray crystallography. We obtained partial knockout mutants for TcCS by using CRISPR/Cas9. TcCS-knocked out epimastigotes showed a lower proliferation rate and a diminished resistance to short-time (30 min) exposition to 120 uM H2O2 when compared with the control (a lineage constitutively expressing Cas9). When these parasites were submitted to nutritional stress in the presence (or not as a control) of 5 mM L-Serine, 5 mM OAS different concentrations (0.2, 0.4 or 1 mM) of L-Cys we observed that: i. L-Cys concentrations over of 200 mM were lethal to the mutants after 48 hours and; ii. L-Ser and OAS contributed to the survival of both, mutants and wild type parasites to severe starvation. We found that DTcCS parasites had their ability to differentiate to metacyclic trypomastigotes diminished. When the capacity of these metacyclic trypomastigotes to infect mammalian host cells was assayed, we observed an over rate of infection after 48 hours and an increase of trypomastigote bursting. However, we noticed severe morphological alterations in the released trypomastigotes when compared to controls. Altogether, these data indicate that cysteine has an important role during epimastigotes proliferation, metacyclogenesis and the infection of mammalian cells, which could prompt this enzyme as a possible drug target. 
