NEK kinase TcRDK2 controls differentiation, host-cell infection, and remodeling of the translation initiation machinery in Trypanosoma cruzi

Trypanosoma cruzi, the etiologic agent of Chagas disease, alternates between replicative epimastigotes and amastigotes and non-dividing, mammal-infective metacyclic and bloodstream trypomastigotes. Protein phosphorylation is a major regulatory mechanism in trypanosomatids, whose kinomes reveal an expanded family of NIMA-related kinases (NEKs). Here, we investigated the role of T. cruzi RDK2 (Repressor of Differentiation Kinase 2), a conserved NEK that carries a C-terminal pleckstrin homology (PH) domain. Endogenous gene tagging showed that TcRDK2 is expressed in all major life-cycle stages and displays a cytoplasmic distribution. CRISPR/Cas9-mediated knockout of TcRDK2 did not markedly alter epimastigote growth in rich medium but caused a significant accumulation of cells with abnormal nuclear/kinetoplast configurations, consistent with defects in kinetoplast segregation and cytokinesis; TcRDK2-null parasites also showed reduced in vitro metacyclogenesis and failed to establish efficient infections in human fibroblasts. To probe gain-of-function effects, we generated tetracycline-inducible overexpression lines for full-length TcRDK2 (RDK2WT), a PH-deleted variant (RDK2{Delta}PH), and a catalytic-dead mutant (RDK2K70A). Overexpression of RDK2WT or RDK2{Delta}PH decreased epimastigote growth, enhanced metacyclogenesis, and strongly impaired host-cell invasion and intracellular amastigote proliferation, with more pronounced phenotypes for RDK2{Delta}PH, suggesting that the PH domain normally restrains TcRDK2 activity in vivo. Phosphoproteomic profiling of RDK2WT-overexpressing epimastigotes identified candidate TcRDK2 substrates and pathways, including translation initiation and cytoskeletal regulation. Together, these data identify TcRDK2 as a NEK kinase that coordinates kinetoplast replication/segregation, metacyclogenesis, and host-cell infection in T. cruzi and support TcRDK2 as a promising, kinetoplastid-specific therapeutic target for Chagas disease.