{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T12:00:53Z","timestamp":1781179253344,"version":"3.54.1"},"posted":{"date-parts":[[2026,6,9]]},"group-title":"Microbiology","reference-count":78,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,9]],"date-time":"2026-06-09T00:00:00Z","timestamp":1780963200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  <jats:italic>Trypanosoma cruzi<\/jats:italic>\n                  , 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\n                  <jats:italic>T. cruzi<\/jats:italic>\n                  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\n                  <jats:italic>TcRDK2<\/jats:italic>\n                  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;\n                  <jats:italic>TcRDK2<\/jats:italic>\n                  -null parasites also showed reduced\n                  <jats:italic>in vitro<\/jats:italic>\n                  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\n                  <jats:sup>WT<\/jats:sup>\n                  ), a PH-deleted variant (RDK2\n                  <jats:sup>\u0394PH<\/jats:sup>\n                  ), and a catalytic-dead mutant (RDK2\n                  <jats:sup>K70A<\/jats:sup>\n                  ). Overexpression of RDK2\n                  <jats:sup>WT<\/jats:sup>\n                  or RDK2\n                  <jats:sup>\u0394PH<\/jats:sup>\n                  decreased epimastigote growth, enhanced metacyclogenesis, and strongly impaired host-cell invasion and intracellular amastigote proliferation, with more pronounced phenotypes for RDK2\n                  <jats:sup>\u0394PH<\/jats:sup>\n                  , suggesting that the PH domain normally restrains TcRDK2 activity in vivo. Phosphoproteomic profiling of RDK2\n                  <jats:sup>WT<\/jats:sup>\n                  -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\n                  <jats:italic>T. cruzi<\/jats:italic>\n                  and support TcRDK2 as a promising, kinetoplastid-specific therapeutic target for Chagas disease.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    Chagas disease, caused by the parasite\n                    <jats:italic>Trypanosoma cruzi<\/jats:italic>\n                    , 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\n                    <jats:italic>T. cruzi<\/jats:italic>\n                    . 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.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.07.730777","type":"posted-content","created":{"date-parts":[[2026,6,9]],"date-time":"2026-06-09T15:14:55Z","timestamp":1781018095000},"source":"Crossref","is-referenced-by-count":0,"title":["NEK kinase TcRDK2 controls differentiation, host-cell infection, and remodeling of the translation initiation machinery in\n                  <i>Trypanosoma cruzi<\/i>"],"prefix":"10.64898","author":[{"given":"Juliana N.","family":"Roson","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221-006, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica","family":"Huckleberry","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221-006, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2178-381X","authenticated-orcid":false,"given":"Noelia","family":"Lander","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221-006, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5581-9027","authenticated-orcid":false,"given":"Miguel A.","family":"Chiurillo","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221-006, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026061104300544000_2026.06.07.730777v1.1","doi-asserted-by":"crossref","unstructured":"Beatty NL , Hamer GL , Moreno-Peniche B , Mayes B , Hamer SA . 2025. Chagas Disease, an Endemic Disease in the United States. Emerg Infect Dis 31.","DOI":"10.3201\/eid3109.241700"},{"key":"2026061104300544000_2026.06.07.730777v1.2","doi-asserted-by":"crossref","first-page":"e132","DOI":"10.1017\/S095026882510071X","article-title":"Endemic potential of Chagas disease in the U.S. southwest: updated surveillance of infected Triatomines from the U.S.-Mexico border region","volume":"153","year":"2025","journal-title":"Epidemiol Infect"},{"key":"2026061104300544000_2026.06.07.730777v1.3","unstructured":"Chagas disease. https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/chagas-disease-(american-trypanosomiasis). 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