Role of Alanine Racemase and Free D- Alanine In Trypanosoma cruzi

GIRARD, RM; PAES, LS; PEREIRA, CA; DA SILVA, MS; PAVANI, RS; ELIAS, MC; SILBER, ARIEL

Caxambu

Congreso; XLIII Annual Meeting on Basic Research in Chagas? Disease; 2016

SBPz

L-amino acids has been shown to be relevant in many Trypanosoma cruzi biological processes.However, little is known about the functions of D-amino acids on the biology of the parasite. Theproline racemase (PR) was firstly describe in T. cruzi, as a potent mitogen. PR is also involvedin the infectivity of the host cells by the parasite. The occurrence of D-Alanine and an AlanineRacemase (AR) activity was also described in Leishmania amazonensis. We identified by thefirst time in a pathogenic protozoa a gene encoding a putative AR (TcAR), a pyridoxal 5?-phosphate dependent enzyme that catalyzes the racemization between L-and D-alanine. Wecloned and expressed TcAR and characterized its recombinant product (rTcAR). rTcAR showeda MW of 43kDa, and Vmax and Km values of 21.5 ± 6.04 mM and 60 ± 6.7 μmol/min/mgrespectively. Immunofluorescence with a specific anti-rTcAR serum showed a cytoplasmiclocalization for TcAR. Specific activity was higher in insect stages compared to mammalianstages. Interestingly, both D- and L-alanine were able to maintain the parasites viability undermetabolic stress conditions. In addition, we showed that D-Alanine is a substrate of an ATPdependentalanine ligase and is excreted in the extracellular medium. We also selected an ARinhibitor, C3, which was able to reduce rTcAR activity, epimastigotes growth, as well as todisrupt plasma membrane, to induce DNA damage, deregulation of ROS, cytosolic Ca2+ andmitochondrial membrane potential in epimastigote. Furthermore, C3 impaired the intracellularlife cycle of T. cruzi with a high selectivity index (