L. (Leishmania) infantum RPV (MHOM/BR/2002/LPC-RPV) promastigotes were grown at 26 °C in M199 medium (Gibco) supplemented with 40 mM HEPES (pH 7.4), 5 μg/mL hemin, 2 μg/mL biopterin, 1 μg/mL biotin, 2 mM L-glutamine, 500 U/mL penicillin, 50 μg/mL streptomycin, and 10% inactivated fetal bovine serum . Cultures were maintained by performing two weekly passages in which 1 × 106 parasites were inoculated in 5 mL of medium. All experiments were performed using promastigotes in the logarithmic growth phase unless otherwise stated.
The FeSOD-A (LINF_080007900) knockout attempt was initially performed by two rounds of gene replacement by homologous recombination using neomycin phosphotransferase (NEO) and hygromycin phosphotransferase (HYG) as selectable markers . The homology arms used for homologous recombination flank the FeSOD-A coding sequence and are 518 bp long in the 5′UTR and 459 bp long in the 3′UTR. Other knockout attempts were made using the CRISPR/Cas9 system.
The first attempt to knock out FeSOD-A using CRISPR was performed according to Zhang et al. . The single-guide RNAs (sgRNAs) were selected using the Eukaryotic Pathogen CRISPR gRNA Design Tool (http://gRNA.ctegd.uga.edu), and the protospacer sequences were ligated into the pSPneoHHsgRNAaH vector that had been previously digested with the restriction enzyme BbsI (New England Biolabs). Parasites containing the plasmid pLPhygCas9 were independently transfected with two different versions of the plasmid pSPneoHHsgRNAaH, containing sgRNAs 144 or 282, and a donor DNA to insert stop codons within the FeSOD-A coding sequence.
A second attempt to generate FeSOD-A null mutants using CRISPR/Cas9 was performed as previously described by Beneke et al. . The plasmid pTB007, which has hygromycin as a resistance marker, was used to express SpCas9 and T7RNAP. Parasites carrying this plasmid and successfully expressing Cas9 were transfected with donor DNAs and templates for guide RNAs chosen with the LeishGEdit tool. Plasmids pTNeo v1 and pTBlast v1 were used for PCR amplification of the donors with 30 bp-long homology arms. sgRNA templates were generated by PCR using the G00 primer. All primers used to construct the plasmids and DNA fragments are listed in Additional file 1: Table S1.
All transfections were performed as previously described . The selection of Leishmania clones was done by plating the parasites in semi-solid M199 medium, and selective drugs were added as per the resistance markers carried by the mutants as follows: 40 µg/mL G418 (Gibco), 400 µg/mL hygromycin B (Invitrogen), or 10 µg/mL blasticidin (Gibco). After that, selection drugs were used in the weekly passages of the cultures, but all experiments to assess the parasites' phenotype were conducted in the absence of selection drugs.
Deletion assessment was performed by PCR using primers to amplify the FeSOD-A coding sequence and primers to evaluate the replacement of FeSOD-A alleles by resistance marker sequences (Additional file 1: Table S1). Protein levels were assessed by western blotting using the polyclonal antibody anti-FeSOD-A  at a dilution of 1:500. Densitometric analysis was performed to compare FeSOD-A levels with the α-tubulin reference.
To evaluate the growth of the parasite, 1 × 106 promastigotes/mL were inoculated in M199 medium, and the parasite concentration was determined daily using the Z1 Coulter Counter (Beckman Coulter) cell counter.
To evaluate the parasite’s susceptibility to antimony, AMB, miltefosine, and menadione, 2 × 106 promastigotes were incubated in 1 mL M199 medium containing various concentrations of drugs. The number of parasites grown in the absence and presence of the drug after 48 h of incubation was determined using the Z1 Coulter Counter (Beckman Coulter). The 50% growth inhibitory concentration (IC50) was determined using the non-linear regression–variable slope model as per the equation "log (inhibitor) vs. response" in GraphPad Prism v.8.2.0.
To assess transcript levels, quantitative reverse-transcription PCR (RT-qPCR) analysis was performed using the cDNA of wild-type (WT) parasites and mutants. Promastigotes (approximately 108 cells) were harvested and resuspended in 1 mL TRIzol Reagent (Invitrogen), and total RNA was extracted using the chloroform method. The RNA was treated with DNase I (Ambion), and the cDNA was obtained using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. All cDNA samples were diluted to 100 ng/μL and used in the RT-qPCR amplification reaction performed using 1X SYBR GREEN master mix (Applied Biosystems) and the specific primers listed in Additional file 1: Table S1. Specific primers for each enzyme were designed using conserved nucleotide regions to amplify the different gene copies found in TritrypDB (tritrypdb.org). The levels of transcripts of the following enzymes were evaluated: six FeSODs (SODA [LINF_080007900], putative SODB1 [LINF_320024000], SODB2 [LINF_320024100], SOD putative [LINF_300033000], SOD putative [LINF_320033200], and SOD putative [LINF_340012900]) and six enzymes from the antioxidant defence system (APX [LINF_340005600], NADH-dependent fumarate reductase [FRD; LINF_350013000, LINF_350016600, and LINF_350016700], TXNPx [LINF_150018600, LINF_150018800, and LINF_150019000], type II GPX-like TXNPx [LINF_260013100], GPX putative [LINF_360038100], and PRX [LINF_230005400]). The DNA polymerase gene (LINF_160021500) was used as a constitutive normaliser. Amplifications were performed using a QuantStudio™ 12 K Flex system (Thermo Fisher Scientific). The PCR conditions were as follows: initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 60 °C for 30 s. Fluorescence was measured after each cycle. Transcript levels were determined using the comparative CT method (2−ΔΔCT method).
Cells derived from the human monocytic strain THP-1 were cultured in complete Rowell Park Memorial Institute (RPMI)-1640 medium (supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin). Monocytes were differentiated into macrophages by the addition of 20 ng/mL phorbol myristate acetate (PMA). After 72 h, the macrophages were infected with Leishmania cultures on the second day of the stationary phase (10 parasites per macrophage) for 6 h. The parasites that failed to infect the macrophages were washed away, and the infected macrophages were incubated for 72 h in RPMI-1640 medium. The infectivity of mutant clones was assessed immediately after the conclusion of the 6 h incubation period as well as after 72 h. The slides were stained with rapid panoptic (Laborclin) and photographed, and the infection was quantified by counting intracellular amastigotes using ImageJ free software.
For all experiments, at least three technical replicates were performed for each of the three biological replicates. Data were analysed using GraphPad Prism v.8.2.0. Statistical significance was set at p < 0.05. The p-values were reported as per the GraphPad Prism format, where ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), and **** (p ≤ 0.0001).