B Cyclic AMP binding proteins in Trypanosoma cruzi J

JOSE ESCALONA; GUILLERMO DI MARIO ; GABRIEL FERRI; MARTIN EDREIRA

Congreso; Molecular Parasitology Meeting XXXI; 2021

Cyclic AMP signaling have shown to be involved in Trypanosoma cruzi biology. However, little is known about the downstream effectors in thispathway in the parasite. Previous reports showed that at the genomic level this parasite has several proteins that possess cyclic nucleotide binding domain (CNB),such as Protein kinase A (PKA). Although, it has also been published that, unlike its mammalian counterpart, the trypanosomatid PKA does not bind cAMP. Inorder to increase the knowledge about the cAMP mediated pathway in T. cruzi, we have cloned and biochemically characterized several cAMP binding proteins inT. cruzi. At the bioinformatic level, several proteins were found in the parasite?s genome. Six candidates, TcCLB.418221.20, TcCLB.504153.20, TcCLB.504449.30,TcCLB.508523.80, TcCLB.510691.30 and TcCLB.508273.30 were selected to perform docking models to evaluate cAMP binding capability in silico. Thesecandidates then were cloned and expressed with his-tag or GST-tag in E. coli. Bacterially expressed proteins and a cAMP-agarose resin were used in pull downand displacement experiments, in order to confirm cAMP binding. Interaction assays were then performed using trypomastigote lysates obtained from infectedVero cell cultures. The eluted proteins were analyzed by mass spectrometry obtaining potential partners candidates, that will be further studied.