Proximity-dependent biotinylation and identification of flagellar proteins inTrypanosoma cruzi

Abstract

The flagellated kinetoplastid protozoan and causative agent of human Chagas disease,Trypanosoma cruzi, inhabits both invertebrate and mammalian hosts over the course of its complex life cycle. In these disparate environments,T. cruziuses its single flagellum to propel motile life stages and in some instances, to establish intimate contact with the host. Beyond its role in motility, the functional capabilities of theT. cruziflagellum have not been defined. Moreover, the lack of proteomic information for this organelle, in any parasite life stage, has limited functional investigation. In this study, we employed a proximity-dependent biotinylation approach based on the differential targeting of the biotin ligase, TurboID, to the flagellum or cytosol in replicative stages ofT. cruzi, to identify flagellar-enriched proteins by mass spectrometry. Proteomic analysis of the resulting biotinylated protein fractions yielded 218 candidate flagellar proteins inT. cruziepimastigotes (insect stage) and 99 proteins in intracellular amastigotes (mammalian stage). Forty of these flagellar-enriched proteins were common to both parasite life stages and included orthologs of known flagellar proteins in other trypanosomatid species, proteins specific to theT. cruzilineage and hypothetical proteins. With the validation of flagellar localization for several of the identified candidates, our results demonstrate that TurboID-based proximity proteomics is an effective tool for probing subcellular compartments inT. cruzi. The proteomic datasets generated in this work offer a valuable resource to facilitate functional investigation of the understudiedT. cruziflagellum.

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance inT. brucei

“…CARP3 differentially regulates the abundance of ACs, and a model predicts that it controls the relative abundance of functionally different cyclases for integration of various external signals (Bachmaier et al ., 2022). The flagellar tip and membrane localization of CARP3 was also seen in T. cruzi (Won et al ., 2023). The specific role in signalling from the environment to the cell, e.g.…”

Gene editing of putative cAMP and Ca2+‐regulated proteins using an efficient cloning‐free CRISPR/Cas9 system in Trypanosoma cruzi

“…Interestingly, in a proximity‐dependent biotinylation approach of flagellar proteins of T . cruzi , unlike other FLAM proteins (FLAM1,2,3,5,7 and 8), TcFLAM6 was only detected in epimastigotes and not in intracellular amastigotes (Won et al, 2023).…”

Gene editing of putative cAMP and Ca2+-regulated proteins using an efficient cloning-free CRISPR/Cas9 system inTrypanosoma cruzi

“…Moreover, TcFLAM6-KO epimastigotes exhibited a lower growth rate in the exponential phase compared to parental cells, which in addition to the unsuccessful generation of a null mutant in a first round of transfection with two resistance markers, confirms the importance of this gene in T. cruzi epimastigotes. Interestingly, in a proximity-dependent biotinylation approach of flagellar proteins of T. cruzi, unlike other FLAM proteins (FLAM1,2,3,5,7 and 8), TcFLAM6 was only detected in epimastigotes and not in intracellular amastigotes (Won et al, 2023). CC2CP predicted CBDs were previously analyzed in silico in a bioinformatic identification of cyclic nucleotide binding proteins in T. cruzi (Jager et al, 2014).…”