Kharon Is Crucial for <i>Trypanosoma cruzi</i> Morphology but Does Not Impair In Vitro Infection
Chagas disease, caused by <i>Trypanosoma cruzi</i>, is a neglected tropical disease with few options for treatment and no available vaccine. Deletion mutants for live attenuated vaccines, particularly deletions of proteins related to the cytoskeleton, have been widely tested in related p...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-03-01
|
| Series: | Pathogens |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-0817/14/4/312 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850180708310450176 |
|---|---|
| author | Jose Luis Saenz-Garcia Normanda Souza-Melo Juliana Severo Miranda Beatriz Borges Lisandro A. Pacheco-Lugo Jose M. Quintero-Solano Nilmar Moretti Richard Wheeler Lia C. Soares-Medeiros Wanderson D. DaRocha |
| author_facet | Jose Luis Saenz-Garcia Normanda Souza-Melo Juliana Severo Miranda Beatriz Borges Lisandro A. Pacheco-Lugo Jose M. Quintero-Solano Nilmar Moretti Richard Wheeler Lia C. Soares-Medeiros Wanderson D. DaRocha |
| author_sort | Jose Luis Saenz-Garcia |
| collection | DOAJ |
| description | Chagas disease, caused by <i>Trypanosoma cruzi</i>, is a neglected tropical disease with few options for treatment and no available vaccine. 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 <i>T. cruzi</i>. Kharon is one such protein, identified as being associated with the cytoskeleton in <i>Leishmania</i> and essential for amastigote replication. Here we investigated the <i>T. cruzi</i> Kharon ortholog (<i>Tc</i>Kharon) to test if it has orthologous function and thus potential in generating a live attenuated vaccine. In silico analysis predicted <i>Tc</i>Kharon to be an intrinsically disordered protein, consistent with its ortholog feature, and GFP fusion protein revealed that <i>Tc</i>Kharon 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, <i>Tc</i>Kharon<sup>−/−</sup> mutants retained the ability to differentiate into metacyclic trypomastigotes and exhibited in vitro infection rates comparable to wild-type parasites. Our data show that <i>Tc</i>Kharon is crucial for cell morphology. However, in contrast to close related parasites, <i>Tc</i>Kharon is not essential for in vitro infectivity. |
| format | Article |
| id | doaj-art-2b457f65bf864a368c28d11d4887e68e |
| institution | OA Journals |
| issn | 2076-0817 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pathogens |
| spelling | doaj-art-2b457f65bf864a368c28d11d4887e68e2025-08-20T02:18:04ZengMDPI AGPathogens2076-08172025-03-0114431210.3390/pathogens14040312Kharon Is Crucial for <i>Trypanosoma cruzi</i> Morphology but Does Not Impair In Vitro InfectionJose Luis Saenz-Garcia0Normanda Souza-Melo1Juliana Severo Miranda2Beatriz Borges3Lisandro A. Pacheco-Lugo4Jose M. Quintero-Solano5Nilmar Moretti6Richard Wheeler7Lia C. Soares-Medeiros8Wanderson D. DaRocha9Laboratório de Genômica Funcional de Parasitos (GFP), Universidade Federal de Paraná, Curitiba 81531-980, BrazilLaboratório de Ultraestrutura Hertha Mayer, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21491-590, BrazilLaboratório de Genômica Funcional de Parasitos (GFP), Universidade Federal de Paraná, Curitiba 81531-980, BrazilLaboratório de Biologia Celular, Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba 81310-020, BrazilFacultad de Ciencias Básicas y Biomédicas, Universidad Simón Bolívar, Barranquilla 080020, ColombiaLaboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genόmica, Instituto Politécnico Nacional, Reynosa 88710, MexicoLaboratório de Biologia Molecular de Patógenos (LBMP), Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04023-062, BrazilNuffield Department of Medicine, University of Oxford, Oxford OX1 3SY, UKLaboratório de Biologia Celular, Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba 81310-020, BrazilLaboratório de Genômica Funcional de Parasitos (GFP), Universidade Federal de Paraná, Curitiba 81531-980, BrazilChagas disease, caused by <i>Trypanosoma cruzi</i>, is a neglected tropical disease with few options for treatment and no available vaccine. 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 <i>T. cruzi</i>. Kharon is one such protein, identified as being associated with the cytoskeleton in <i>Leishmania</i> and essential for amastigote replication. Here we investigated the <i>T. cruzi</i> Kharon ortholog (<i>Tc</i>Kharon) to test if it has orthologous function and thus potential in generating a live attenuated vaccine. In silico analysis predicted <i>Tc</i>Kharon to be an intrinsically disordered protein, consistent with its ortholog feature, and GFP fusion protein revealed that <i>Tc</i>Kharon 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, <i>Tc</i>Kharon<sup>−/−</sup> mutants retained the ability to differentiate into metacyclic trypomastigotes and exhibited in vitro infection rates comparable to wild-type parasites. Our data show that <i>Tc</i>Kharon is crucial for cell morphology. However, in contrast to close related parasites, <i>Tc</i>Kharon is not essential for in vitro infectivity.https://www.mdpi.com/2076-0817/14/4/312<i>Trypanosoma cruzi</i>morphologyCRISPR/Cas9 |
| spellingShingle | Jose Luis Saenz-Garcia Normanda Souza-Melo Juliana Severo Miranda Beatriz Borges Lisandro A. Pacheco-Lugo Jose M. Quintero-Solano Nilmar Moretti Richard Wheeler Lia C. Soares-Medeiros Wanderson D. DaRocha Kharon Is Crucial for <i>Trypanosoma cruzi</i> Morphology but Does Not Impair In Vitro Infection Pathogens <i>Trypanosoma cruzi</i> morphology CRISPR/Cas9 |
| title | Kharon Is Crucial for <i>Trypanosoma cruzi</i> Morphology but Does Not Impair In Vitro Infection |
| title_full | Kharon Is Crucial for <i>Trypanosoma cruzi</i> Morphology but Does Not Impair In Vitro Infection |
| title_fullStr | Kharon Is Crucial for <i>Trypanosoma cruzi</i> Morphology but Does Not Impair In Vitro Infection |
| title_full_unstemmed | Kharon Is Crucial for <i>Trypanosoma cruzi</i> Morphology but Does Not Impair In Vitro Infection |
| title_short | Kharon Is Crucial for <i>Trypanosoma cruzi</i> Morphology but Does Not Impair In Vitro Infection |
| title_sort | kharon is crucial for i trypanosoma cruzi i morphology but does not impair in vitro infection |
| topic | <i>Trypanosoma cruzi</i> morphology CRISPR/Cas9 |
| url | https://www.mdpi.com/2076-0817/14/4/312 |
| work_keys_str_mv | AT joseluissaenzgarcia kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection AT normandasouzamelo kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection AT julianaseveromiranda kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection AT beatrizborges kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection AT lisandroapachecolugo kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection AT josemquinterosolano kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection AT nilmarmoretti kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection AT richardwheeler kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection AT liacsoaresmedeiros kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection AT wandersonddarocha kharoniscrucialforitrypanosomacruziimorphologybutdoesnotimpairinvitroinfection |