Fig 1.
Bayesian phylogram inferred from PGFS sequences from different T. cruzi strains.
Fig 2.
Characterization of T. cruzi ΔPGFS mutant clones and PGFS add-backs.
Screening of T. cruzi PGFS knockout clones after CRISPR/Cas9 with sgRNA187 was performed by both PCR and western blot. (A) Schematic representation of PGFS sequence before and after the insertion of donor DNAs with stop codons and XhoI restriction site; (B) The complete PGFS coding sequences (1140 bp) were amplified by PCR. The primers can successfully amplify both the WT sequence and the mutant sequences containing stop codons and the XhoI restriction site. The PCR products were purified and digested with the restriction enzyme XhoI. 3.5 μg of DNA was applied per well. The WT sequence does not have the XhoI site and wasn’t digested. On the other hand, the mutant sequences edited were cleaved by the XhoI restriction enzyme and produced two fragments, one with 216 bp and another with 941 bp. (C) Western blotting shows the deletion of the PGFS gene after CRISPR and also the expression of PGFS in the add-back parasites. The western blots are performed using polyclonal anti-PGFS antibody and monoclonal anti-α-tubulin as a normaliser. The fold-change was calculated using the WT parasites as standards. WT, wild-type; c27 and c29, mutant clones; MW, molecular weight; CN, negative control; bp, base pair; KDa, kiloDalton.
Fig 3.
Drug susceptibility of ΔPGFS mutant parasites.
Parasites were cultured in the presence of different concentrations of (A) benznidazole (1.25 to 15 μM) and (B) nifurtimox (0.3125 to 5 μM). Their growth was determined after 7 days of incubation with or without the drugs. Data represent the mean with standard deviations of three independent experiments performed in triplicate. The IC50 was determined through the nonlinear regression—variable slope model, using the "log (inhibitor) vs. response" equation in GraphPad Prism v.8.2.0. A Two-way ANOVA test with Bonferroni post hoc test was used to compare WT parasites and mutants for each drug concentration. * represents significant differences between the WT and the ΔPGFS clone c27 (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p ≤ 0.0001). + represents significant differences between the WT and the ΔPGFS clone c29 (+ p < 0.05; ++ p < 0.01; +++ p < 0.001; ++++ p ≤ 0.0001). # represents significant differences between the WT and the parasites expressing Cas9 (# p < 0.05; ## p < 0.01; ### p < 0.001; #### p ≤ 0.0001). (C) Comparison of NTR-1 transcription levels between the control parasite (Cas9) and ΔPGFS mutants. (D) Comparison of NTR-1 transcription levels between sensitive parasites (17WTS) and parasites whose resistance to benznidazole was induced in vitro (17LER). The housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HGPRT), was used as a constitutive normalizer and the fold-change was calculated by the 2–ΔΔCt method. An Ordinary one-way ANOVA test with a Bonferroni post hoc test was used to compare WT parasites and mutants. * represents significant differences in relation to the control parasite (Cas9 or 17WTS) (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p ≤ 0.0001).
Fig 4.
Tolerance of ΔPGFS mutant parasites to oxidative stress.
Parasites were cultured in the presence of different concentrations of menadione (1.0 to 4 μM). Their growth was determined after 7 days of incubation with or without the drugs. (A) Comparison between the WT, control parasites expressing Cas9, ΔPGFS c27, and c27 + add-back parasites. (B) Comparison between the WT, control parasites expressing Cas9, ΔPGFS c29, and c29 + add-back parasites. Data represent the mean with standard deviations of three independent experiments performed in triplicate. The IC50 was determined through the nonlinear regression—variable slope model, using the "log (inhibitor) vs. response" equation in GraphPad Prism v.8.2.0. A Two-way ANOVA test with Bonferroni post hoc test was used to compare WT parasites and mutants for each drug concentration. * represents significant differences between the WT and the ΔPGFS clone c27, or between the WT and the ΔPGFS clone c29 (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p ≤ 0.0001). # represents significant differences between the WT and the c27 + add-back, or between the WT and the c29 + add-back (# p < 0.05; ## p < 0.01; ### p < 0.001; #### p ≤ 0.0001).
Fig 5.
Evaluation of lipid body in ΔPGFS mutant parasites through Nile Red staining.
The lipid droplets within the parasites were visualized and quantified using Nile Red reagent. (A) Representative images of the control parasites expressing Cas9, ΔPGFS clones, and add-back parasites at three distinct growth stages at three distinct growth stages: log, logarithmic phase (after 48 hours); s1, stationary phase 1 (after 7 days); s2 stationary phase 2 (after 15 days). (B) Geometric mean fluorescence in FL1 channel in arbitrary units comparing the quantity of lipid body vesicles in Cas9-expressing control parasites, PGFS clones, and add-back parasites at three distinct growth stages. A One-way ANOVA test with Bonferroni post hoc test was used to compare control parasites and mutant clones. *represents significant differences compared to the control parasite (Cas9) (* p < 0.05; ** p < 0.01; *** p < 0.001).
Fig 6.
Infectivity ΔPGFS mutant parasites.
L929 fibroblasts were infected with trypomastigote forms of control parasites expressing Cas9, ΔPGFS clones, and add-back parasites at a ratio of 1:10. Data represent the mean with standard deviations of three independent experiments performed in sextuplicate. (A) The graph shows the number of intracellular amastigotes per 100 macrophages 48h after infection. (B) The graph shows the percentage of infected fibroblasts. A One-way ANOVA test with Bonferroni post hoc test was used to compare parasites expressing Cas9 and mutant clones. *represents significant differences compared to the control parasite (Cas9) (* p < 0.05; ** p < 0.01; *** p < 0.001).