*3.4. The Nitrosative Challenge Negatively Modulated Antioxidant Proteins of NO-Susceptible L. braziliensis Strain*

As the 2853 and 2856 strains have different responses to pro-oxidant and antioxidant stimuli and exhibit significant differences in their proteomes, we analyzed the abundance levels of proteins involved in the response to oxidative stress and in the maintenance of parasites' redox homeostasis in our proteomics dataset. First, we observed that cumulative concentration of proteins involved in those processes is significantly higher in NO-resistant parasites than in the NO-susceptible. Interestingly, upon nitrosative challenge, the resistant 2853 strain maintains higher concentrations of those proteins, while the susceptible 2856 exhibits a significant decrease of them (Figure 4A).

**Figure 3.** NO-resistance modulates protein abundance of *L. braziliensis* strains. Whole cell lysates from 2853, 2853+NO (2853 challenged with the 1/5 IC50/4 h NaNO2), 2856, and 2856+NO (2856 challenged with the 1/5 IC50/4 h NaNO2) strains were processed by MED-FASP and analyzed by LC-MS/MS. Protein abundances were calculated based on the raw spectral intensities. (**A**) Total protein content per cell of four biological replicates for each strain. Graphs represent mean ± SD of four independent experiments. Statistical differences by *t* test (\*\*\*\* *p* < 0.0001). (**B**) Violin plots depicting distribution of log10 transformed protein concentration values of proteins identified and quantified in each strain. Protein names above violin plots represent most abundant proteins common for all strains. (**C**) Principal component analysis of the absolute protein concentration values of all quantified proteins determined by the Total Protein Approach (TPA) method.

We also analyzed the concentration of specific proteins that are directly involved in the response to oxidative stress. The abundance of ascorbate peroxidase (APx), a heme-containing enzyme that catalyzes the conversion of H2O2 into water, was oppositely modulated by NO challenge, increasing in the 2853 strain and decreasing in the 2856 (Figure 4B). The NO-susceptible strain challenged with NaNO2 also exhibited a significant decrease in the absolute concentration of enzymes related to trypanothione-dependent hydroperoxide metabolism, such as tryparedoxin 1 (TXN1) (Figure 4C), tryparedoxin peroxidase (TXNPx) (Figure 4D), and trypanothione reductase (TR) (Figure 4E). Despite the impairment of TXNPx and TR abundance caused by nitrosative stress in NO-resistant parasites, the concentration of these proteins was higher than that observed in the NOsusceptible ones challenged with NO donor (Figure 4D,E). Interestingly, in conditions without NO challenge, NO-resistant strain also exhibited significant higher concentration of superoxide dismutase (SOD) when compared to the NO-susceptible one; however, treatment with NaNO2 significantly decreased cumulative SOD concentration to levels

detected in 2856 strain (Figure 4F). These results show that upon NO challenge, the NOresistant strain is more able to sustain the levels of trypanothione system's enzymes than the NO-susceptible strain.

**Figure 4.** The antioxidant proteins of NO-susceptible *L. braziliensis* strain are negatively modulated after the nitrosative challenge. (**A**) Cumulative concentration of all identified proteins involved in biological processes of "*cell redox homeostasis and response to oxidative stress*". Each symbol shows the total sum of the concentration values of proteins involved in those processes in each of the four biological replicates. Concentrations of specific proteins involved in response to oxidative stress: (**B**) ascorbate peroxidase (APx); (**C**) tryparedoxin 1 (TXN1); (**D**) tryparedoxin peroxidase (TXNPx); (**E**) trypanothione reductase (TR); and (**F**) superoxide dismutase (SOD). 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.
