Revealing the biological role of the Trypanosoma cruzi Alanine racemase using CRISPR-Cas9

GIRARD, RM; PAES, LS; CRISPIM, M; ALENCAR, MB; CUEVAS, R; PEREIRA, CA; SILBER, ARIEL

Evento virtual

Jornada; Molecular Parasitology Meeting XXXI, Genetic Society of America; 2020

Genetic Society of America

In Trypanosoma cruzi, the etiological agent of Chagas?s disease, amino acids participate of several critical processes in the parasite biology, such asosmoregulation, cell differentiation and host cell invasion. Some of them provide reducing power for mitochondrial ATP synthesis. We recently demonstratedthat L-Ala, can be a metabolic waste of glycolysis, and can be a nutrient which can be taken up to fuel the oxidative phosphorylation. In this work we show that T.cruzi encodes a functional Alanine Racemase (TcAR). The enzyme was cloned and expressed and its recombinant version (rTcAR), and was biochemicallycharacterized. Km and Vmax were determined for the reaction in both directions (21.5 ± 6.04 mM and 65 ± 9.4 μmol/min/mg (D to L) and 26.1 ± 7.9 mM and 53 ±6.3 μmol/min/mg (L to D)). Additionally, we showed that D-Ala can be taken up by epimastigotes through a low specificity non-stereoselective active transportsystem. D-Ala uptake happens with a Vmax of 1.14 ± 0.75 nmol·min−1 per 2 × 107 and a Km of 1.46 ± 1.4 mM. The incorporated D-Ala can be completely oxidizedto CO2, supplying electrons for the oxidative phosphorylation. Of note, D-Ala is not transaminated by the parasite transaminases. These data suggest that the DAlaoxidation mainly occurs through the TcAR. To verify this hypothesis, we generated a lineage of T. cruzi CL Brener hemi-knockout for TcAR (TcAR-/+) usingCRISPR-Cas9 technology, and we characterised the phenotype of the TcAR-/+ parasites. TcAR-/+ epimastigotes, when compared to Cas9-expressing controls,showed a decrease in their proliferation rate and an increased sensitivity to nutritional stress. As expected, TcAR-/+ epimastigotes showed a diminished vialbilityin presence of D-Ala, when compared to controls. Additionally, TcAR-/+ epimastigotes incubated with [14C(U)]-D-Ala, exhibited a decrease of the radiolabelledCO2 compared to the control cell lines, indicating the successful impairment of the D-Ala degradation pathway. Our results point that TcAR is the key enzyme forD-Ala oxidation and appears to be involved in other biological process such as resistance to nutritional stress and parasite replication. To conclude, themetabolism of DL-Ala and the related AR activity underlines the outstanding metabolic flexibility and the relevance of the D-amino acids metabolism in theseorganisms.