# Kharon Is Crucial for Trypanosoma cruzi Morphology but Does Not Impair In Vitro Infection

## TL;DR

This study shows that TcKharon is important for the shape of Trypanosoma cruzi but not for its ability to infect cells in the lab.

## Contribution

The study reveals the specific role of TcKharon in T. cruzi morphology and its non-essential role in in vitro infection.

## Key findings

- TcKharon disruption causes morphological defects and impaired proliferation in T. cruzi.
- TcKharon−/− mutants can still differentiate into metacyclic trypomastigotes.
- In vitro infection rates of TcKharon−/− mutants are similar to wild-type parasites.

## Abstract

Chagas disease, caused by Trypanosoma cruzi, 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 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−/− 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.