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

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.

Keywords: CRISPR/Cas9; Trypanosoma cruzi; morphology.

Figure 1

Phylogenetic tree from protein sequences of related trypanosomatids, and TcKharon structure prediction using AlphaFold2. (A) Amino acid sequences from TriTrypDB (https://tritrypdb.org/tritrypdb/app accessed on 26 March 2021) were aligned using the SeaView software. The aligned sequences were used to generate a phylogenetic tree (SeaView 5.4). (B) Prediction model from https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2 (accessed on 26 March 2021). The colors indicate the predicted pLDDT values for TcKharon as given in the key.

Figure 2

Localization of TcKharon tagged with GFP. WT parasites transfected with the plasmid pTREX-GFP (control plasmid—epimastigotes expressing GFP) (A), or pTREX-TcKharon::GFP (epimastigotes expressing TcKharon::GFP) (B). These cells were either treated or not treated with PEME buffer containing TritonX-100 for cytoskeleton extraction as indicated (see Section 2). Left images, DAPI staining (blue); center images, GFP fluorescence (green); and right images, overlay of DAPI, GFP fluorescence, and DIC images.

Figure 3

Genome editing of TcKharon using CRISPR/Cas9. (A) Schematic representation of the sgRNA targeting site by the SaCas9 ribonucleoprotein (RNP) complex in the TcKharon gene (C4B63_14g70). The RNP complex cleaves right after nucleotide 585 of the TcKharon coding sequence (CDS length: 1218 bp). The insertion point of a BglII restriction site (italics) is represented in order to easily track parasite editing through DNA digestion. Stop codons (asterisks) ensure CDS disruption by homologous recombination using a donor DNA sequence. The sequence highlighted in light gray in the donor sequence corresponds to the sgRNA target site, and the dark gray sequence is the PAM sequence. (B) Genotyping of TcKharon showing genome editing of wild-type (WT) parasites. The gels show undigested PCR product (amplicon sizes: WT TcKharon = 1218 bp and TcKharon^−/− = 1235 bp) and BglII-digested (BglII) PCR product of the full-length open reading frame (ORF) of TcKharon of two cultures. The left image corresponds to the PCR product and BglII-digested PCR derived from a mixed population of parasites transfected once with SaCas9 RNP plus donor sequence. The right gel corresponds to the genotyping of a culture transfected twice with RNP complex plus donor DNA. (C) Genotyping of individual clones containing both TcKharon edited alleles.

Figure 4

Growth of TcKharon^−/− is impaired. (A) Parasites were cultured for 7 days in LIT medium and counted daily. Asterisks indicate statistically significant differences between WT and TcKharon^−/− at p < 0.05. TcKharon-AB corresponds to TcKharon^−/− overexpressing TcKharon::GFP. These experiments were performed in triplicate. (B) Quantification of nuclei and kinetoplast numbers through DAPI staining of cells.

Figure 5

TcKharon^−/− epimastigotes are smaller in size. (A) SEM images of T. cruzi WT (a–c), TcKharon^−/− (d–f), and addback (TcKharon-AB) (g–i) cell lines. Central (b,e,h), and right (c,f,i) images correspond to zoom of both ends of the parasites. (B) Scatter plot of cell body area. At least 25 cells were analyzed using Fiji 2.3.0 software (N = 25). Asterisks represent statistically significant differences between groups at *** p < 0.001 and **** p < 0.0001.

Figure 6

The posterior end of TcKharon^−/− epimastigotes is shortened. (A) Immunofluorescence of TcKharon^−/− and WT cells stained with DAPI (blue) and the antibodies to 2F7 (flagellum; red) and α-tubulin (green). (B) Scatter plot showing data from the measures of the distance between the N (Nucleus) and P (Posterior Cortical Tip). The asterisk represents a statistically significant difference at **** p < 0.0001; ns represents a non-statistical difference.

Figure 7

Metacyclogenesis of TcKharon^−/− mutants. (A) Immunofluorescence of metacyclic trypomastigotes (MTs) from WT and TcKharon^−/− cultures showing the defect in morphology. Antibodies to 2F7 (flagellum; red) and actin (green) were used. White arrows indicate the nucleus and kinetoplast. (B) Percentage of metacyclic cells. Asterisk indicates statistically significant difference at p < 0.05. (C) Bar graphs showing the transition of the kinetoplast from the anterior to posterior region of the cell body. (D) Bar graphs showing the number of binucleated cells in the total cell population.

Figure 8

TcKharon disruption does not impair LLC-MK2 cell infection. (A) Tissue culture-derived trypomastigotes (TCTs) of T. cruzi WT, TcKharon^−/− and TcKharon-AB were incubated with LLC-MK2 cells for 5 h. (B) Quantification of amastigotes per cell. The asterisk represents a statistically significant difference at p < 0.05); ns represents a non-statistical difference.