*3.5. NO-Resistant L. braziliensis Strain Challenged with NO Increases the Abundance of Enzymes Involved in the Glutathione Pathway*

Since glutathione (GSH) is involved in the antioxidant response against oxidative and nitrosative stresses in eukaryotes [34,35], we analyzed the abundance levels of proteins involved in its metabolism in our dataset. Interestingly, although differences in glutathione peroxidase (GPx) cumulative abundance levels were detected only in NOsusceptible parasites challenged with NO donor (Figure 5A), the activity of this enzyme was ~7-fold higher in NO-resistant parasites than in NO-susceptible ones. Additionally, after exposure to theNO donor, GPx activity was significantly upregulated in both strains but was significantly higher in NO-resistant strain (Figure 5B). In addition, upon NO challenge, the concentration of glutamate-cysteine ligase (GSH1), which is an important enzyme responsible for the de novo synthesis of GSH, was significantly increased in NO-resistant parasites (Figure 5C). GSH may also interact directly with NO producing S-nitrosoglutathione (GSNO) [36–38], which can be decomposed by GSNO reductases to oxidized glutathione (GSSG). Alcohol dehydrogenase class III enzymes have GSNO reductase activity and function as a protection against nitrosative stress [39,40]. Notably, we found an alcohol dehydrogenase class III significantly decreased in the 2856 NO-susceptible strain challenged with NaNO2, in relation to 2853 treated with NO donor (Figure 5D). Altogether, these results suggest that GSH metabolism may be triggered by NO in *L. braziliensis* strains, especially in NO-resistant ones. In addition, alcohol dehydrogenase class III could also have GSNO reductase activity in *L. braziliensis,* and its downregulation may contribute to the inefficient NO detoxification in NO-susceptible parasites.

**Figure 5.** The NO-resistant strain increases glutathione metabolism enzymes in response to •NO challenge. (**A**) Cumulative concentration of all glutathione peroxidases (GPx) identified in our dataset. Each symbol shows the total sum of the GPx concentration values in each of the four biological replicates. (**B**) GPx activity was measured in parasite homogenates. (**C**) Absolute concentration of glutamate cysteine ligase (GSH1) in each strain. (**D**) Absolute concentration of alcohol dehydrogenase A4HCQ1 in each strain. Graphs represent mean ± SD of four independent experiments. Statistical

differences by Student's *t* test (\* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001). 2853: NO-resistant strain; 2853+NO: NO-resistant strain challenged with 1/5 IC50/4 h NaNO2; 2856: NO-susceptible strain; and 2856+NO: NO-susceptible strain challenged with 1/5 IC50/4 h NaNO2.
