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

ABSTRACT 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 nucleus/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 (RDK2 WT ), a PH-deleted variant (RDK2 ΔPH ), and a catalytically dead mutant (RDK2 K70A ). Overexpression of RDK2 WT or RDK2 ΔPH decreased epimastigote growth, enhanced metacyclogenesis, and strongly impaired host cell invasion and intracellular amastigote proliferation, with more pronounced phenotypes for RDK2 ΔPH , suggesting that the PH domain normally restrains TcRDK2 activity in vivo . Phosphoproteomic profiling of RDK2 WT -overexpressing epimastigotes revealed broad phosphoproteome remodeling associated with TcRDK2 upregulation, including changes affecting proteins linked to translation initiation and the cytoskeleton. Together, these findings support a role for TcRDK2 as a NEK kinase in kinetoplast replication/segregation, metacyclogenesis, and host cell infection in T. cruzi and highlight TcRDK2 as a promising kinetoplastid-specific therapeutic target for Chagas disease. IMPORTANCE Chagas disease, caused by the parasite Trypanosoma cruzi , remains a major health problem with limited treatment options. To persist in both insect vectors and mammalian hosts, the parasite must precisely coordinate cell division, differentiation into infectious forms, and survival inside host cells. Protein kinases are central regulators of these processes and attractive drug targets, yet many remain poorly understood in T. cruzi . In this study, we investigate RDK2, a member of the NIMA-related kinase family. Using gene knockout, inducible overexpression, and global analysis of phosphorylated proteins, we show that RDK2 is required for accurate segregation of mitochondrial DNA, efficient formation of infective insect stage forms, and successful infection and replication in human cells. These findings identify RDK2 as a key regulator that links parasite cell division to infectivity and highlight it as a promising, parasite-specific candidate for future drug development against Chagas disease.