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Background  Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this enzyme is a promising molecular target for drug development against trypanosomatids.    Results  We obtained  Leishmania infantum  mutant clones with lower FeSOD-A expression and investigated their phenotypes. Our attempts to delete this enzyme-coding gene using three different methodologies (conventional allelic replacement or two different CRISPR/methods) failed, as  FeSOD-A  gene copies were probably retained by aneuploidy or gene amplification. Promastigote forms of WT and mutant parasites were used in quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blot analyses, and these parasite forms were also used to assess drug susceptibility. RT-qPCR and western blot analyses revealed that FeSOD-A transcript and protein levels were lower in  FeSOD-A   −/−/ +   L. infantum  mutant clones than in the wild-type (WT) parasite. The decrease in FeSOD-A expression in  L. infantum  did not interfere with the parasite growth or susceptibility to amphotericin B. Surprisingly,  FeSOD-A   −/−/ +   L. infantum  mutant clones were 1.5- to 2.0-fold more resistant to trivalent antimony and 2.4- to 2.7-fold more resistant to miltefosine. To investigate whether the decrease in FeSOD-A expression was compensated by other enzymes, the transcript levels of five FeSODs and six enzymes from the antioxidant defence system were assessed by RT-qPCR. The transcript level of the enzyme ascorbate peroxidase increased in both the  FeSOD-A   −/−/ +  mutants tested. The  FeSOD-A   −/−/ +  mutant parasites were 1.4- to 1.75-fold less tolerant to oxidative stress generated by menadione. Infection analysis using THP-1 macrophages showed that 72 h post-infection, the number of infected macrophages and their intracellular multiplication rate were lower in the  FeSOD-A   −/−/ +  mutant clones than in the WT parasite.    Conclusions  The unsuccessful attempts to delete  FeSOD-A  suggest that this gene is essential in  L. infantum . This enzyme plays an important role in the defence against oxidative stress and infectivity in THP-1 macrophages. FeSOD-A-deficient  L. infantum  parasites deregulate their metabolic pathways related to antimony and miltefosine resistance.    Graphic AbstractSupplementary Information  The online version contains supplementary material available at 10.1186/s13071-021-04838-8.

Downregulation of FeSOD-A expression in Leishmania infantum alters trivalent antimony and miltefosine susceptibility