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Deletion mutants for live attenuated vaccines, particularly deletions of proteins related to the cytoskeleton, have been widely tested in related parasites but candidates have not been tested in T. cruzi. Kharon is one such protein, identified as being associated with the cytoskeleton in Leishmania and essential for amastigote replication. Here we investigated the T. cruzi Kharon ortholog (TcKharon) to test if it has orthologous function and thus potential in generating a live attenuated vaccine. In silico analysis predicted TcKharon to be an intrinsically disordered protein, consistent with its ortholog feature, and GFP fusion protein revealed that TcKharon is associated with the cytoskeleton of epimastigotes. CRISPR-Cas9-mediated gene disruption impaired epimastigote proliferation and cytokinesis, resulting in altered nucleus-to-kinetoplast ratios and pronounced morphological defects, particularly in the posterior cell region. Despite these abnormalities, TcKharon\u2212\/\u2212 mutants retained the ability to differentiate into metacyclic trypomastigotes and exhibited in vitro infection rates comparable to wild-type parasites. Our data show that TcKharon is crucial for cell morphology. However, in contrast to close related parasites, TcKharon is not essential for in vitro infectivity.<\/jats:p>","DOI":"10.3390\/pathogens14040312","type":"journal-article","created":{"date-parts":[[2025,3,25]],"date-time":"2025-03-25T05:35:03Z","timestamp":1742880903000},"page":"312","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Kharon Is Crucial for Trypanosoma cruzi Morphology but Does Not Impair In Vitro Infection"],"prefix":"10.3390","volume":"14","author":[{"given":"Jose Luis","family":"Saenz-Garcia","sequence":"first","affiliation":[{"name":"Laborat\u00f3rio de Gen\u00f4mica Funcional de Parasitos (GFP), Universidade Federal de Paran\u00e1, Curitiba 81531-980, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2025-1088","authenticated-orcid":false,"given":"Normanda","family":"Souza-Melo","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Ultraestrutura Hertha Mayer, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21491-590, Brazil"},{"name":"Laborat\u00f3rio de Biologia Molecular de Pat\u00f3genos (LBMP), Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juliana Severo","family":"Miranda","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Gen\u00f4mica Funcional de Parasitos (GFP), Universidade Federal de Paran\u00e1, Curitiba 81531-980, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2195-1273","authenticated-orcid":false,"given":"Beatriz","family":"Borges","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Biologia Celular, Instituto Carlos Chagas, Funda\u00e7\u00e3o Oswaldo Cruz (Fiocruz), Curitiba 81310-020, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9248-4596","authenticated-orcid":false,"given":"Lisandro A.","family":"Pacheco-Lugo","sequence":"additional","affiliation":[{"name":"Facultad de Ciencias B\u00e1sicas y Biom\u00e9dicas, Universidad Sim\u00f3n Bol\u00edvar, Barranquilla 080020, Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5729-3344","authenticated-orcid":false,"given":"Jose M.","family":"Quintero-Solano","sequence":"additional","affiliation":[{"name":"Laboratorio de Biotecnolog\u00eda Farmac\u00e9utica, Centro de Biotecnolog\u00eda Gen\u03ccmica, Instituto Polit\u00e9cnico Nacional, Reynosa 88710, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0455-2497","authenticated-orcid":false,"given":"Nilmar","family":"Moretti","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Biologia Molecular de Pat\u00f3genos (LBMP), Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard","family":"Wheeler","sequence":"additional","affiliation":[{"name":"Nuffield Department of Medicine, University of Oxford, Oxford OX1 3SY, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9390-9157","authenticated-orcid":false,"given":"Lia C.","family":"Soares-Medeiros","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Biologia Celular, Instituto Carlos Chagas, Funda\u00e7\u00e3o Oswaldo Cruz (Fiocruz), Curitiba 81310-020, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wanderson D.","family":"DaRocha","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Gen\u00f4mica Funcional de Parasitos (GFP), Universidade Federal de Paran\u00e1, Curitiba 81531-980, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"59","DOI":"10.5334\/gh.1150","article-title":"Global, Regional, and National Trends of Chagas Disease from 1990 to 2019: Comprehensive Analysis of the Global Burden of Disease Study","volume":"17","author":"Rojas","year":"2022","journal-title":"Glob. 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