*3.6. Uptake of Glucose Analog and G6PDH Protein Levels Increase in NO-Resistant L. braziliensis Strain after the Nitrosative Challenge*

Because we observed that NO-resistant strain exhibits more robust antioxidant defenses and responds more efficiently to the nitrosative challenge, mainly through the GSH metabolism, we analyzed if proteins involved in GSH reduction are also modified in those parasites, particularly those committed to NADPH production. First, we examined the abundance of enzymes involved in glycolysis and PPP (Figures S3 and S4). Cumulative concentration of glycolytic enzymes was significantly higher in the 2853 strain than in 2856. Resistant strain significantly diminished protein concentration levels of glycolytic enzymes upon NO challenge, but the glycolytic pathway titers remained similar in NOsusceptible parasites after NO exposure (Figure 6A). Notably, cumulative concentration of PPP enzymes was significantly reduced in NO-susceptible parasites but not in NO-resistant ones in response to NO stimulus (Figure 6B). Analysis of phosphotransferase, the first enzyme of the glycolytic pathway, revealed that 2856 strain has higher protein abundance in comparison to 2853; however, after nitrosative challenge, NO-resistant parasites exhibited a significant increase in this enzyme, while the same did not happen in NO-susceptible ones (Figure 6C). Interestingly, such increase is reflected in significantly elevated glucose analogue uptake in those parasites, which was ~2.5-fold higher in 2853 strain than in 2856 after NO exposure (Figure 6D).

Glucose consumed by parasites is rapidly converted to glucose 6-phosphate, which can follow the glycolytic pathway or be driven to PPP and used for maintaining the NADPH pool. In fact, we observed a significant increase in the concentration of G6PDH in the NO-resistant strain upon NO challenge, whereas the enzyme concentration was reduced in the NO-susceptible one under the same stress condition (Figure 6E). In addition, a significant increase in transaldolase (TAL), an enzyme of non-oxidative branch of PPP, was observed in NO-resistant parasites, while a significant reduction of this protein was detected in the NO-susceptible ones (Figure 6F). Analysis of protein abundance of other glycolytic enzymes supports the idea that glucose 6-phosphate may be entering the PPP pathway in NO-resistant parasites (Figures S3 and S4). Particularly, although the levels of 6-phosphofructokinase-1, an important enzyme to glycolytic pathway, were higher in the NO-resistant parasites than in NO-susceptible ones, they were not modulated by the NaNO2 treatment (Figure 6G).
