Generation of T. cruzi strains ectopically expressing proteasome beta 4 subunit variants

PSMB4 TcCLB503891.100 was amplified from T. cruzi CL Brenner genomic DNA using KOD Hot Start DNA Polymerase (EMD Millipore), and sense (5′-AAAGCGGCCGCATGTCGGAGACAACCATTG-3) and antisense (5-CCATGATCTTGATGTAATATAAGGCATTCAGCCCTGCTG-3) primers. The PSMB4^F24L gene was generated from the wild type PSMB4 construct by site-directed mutagenesis using mutagenic sense (5-CAGCAGGGCTGAATGCCTTATATTACATCAAGATCATGG-3′) and antisense (5′-CCATGATCTTGATGTAATATAAGGCATTCAGCCCTGCTG-3′) primers and QuikChange II Site-Directed Mutagenesis Kit (Stratagene). The sequences of the wild type and mutant PSMB4 genes were verified by sequencing and both gene versions were subcloned into the T. cruzi expression vector pTcIndex1 under control of a T7 promoter³⁵. Trypanosoma cruzi CL Brenner epimastigotes were first transfected as described previously³⁶ with the pLEW13 plasmid³⁷ harboring a tetracycline-inducible T7 RNA polymerase gene. Transfected epimastigotes were selected in medium supplemented with neomycin (G418) at 500 μg/ml, and then transfected a second time with either pTcIndex1-PSMB4^wt or pTcIndex1-PSMB4^F24L plasmid. Double transfected epimastigotes were selected in the presence of 500 μg/mL of G418 (Sigma-Aldrich) and 500 μg/mL of hygromycin (Sigma-Aldrich). Susceptibility of double transfected epimastigote cell lines to compounds was assessed using induced (+5 mg/mL of tetracycline) and non-induced parasite cultures after five days of compound treatment. Parasite viability was determined with AlamarBlue (ThermoFisher Scientific).

Reported EC₅₀ values for T. cruzi epimastigotes ectopically expressing PSMB4 proteins were calculated from 3 technical replicates (n= 3; also specified in the Figure 3a caption).