*3.7. Nitrosative Challenge Increases D-Lactate Dehydrogenase Abundance in NO-Resistant L. braziliensis Strain*

As NO challenge seems to positively regulate the first steps of glycolysis, we were interested in analyzing the status of the enzymes at the pathway exit. The concentration levels of pyruvate kinase, the only known regulated glycolytic enzyme in *Leishmania*, did not suffer significant alterations upon exposure to NO (Figure 7A). In addition, the allosteric regulator of pyruvate kinase, 6-phosphofructo-2-kinase, the enzyme responsible for fructose-2,6-bisphosphate biosynthesis, did not suffer significant changes upon exposure to NO (Figure 7B). Intriguingly, we observed a significant increase in the concentration and activity of D-lactate dehydrogenase (D-LDH) in NO-resistant strain upon NO exposure (Figure 7C,D). Indeed, LDH activity was increased 2-fold in NO-resistant parasites after nitrosative challenge and it was 3.3-fold higher in NO-resistant-challenged parasites than in NO-susceptible-challenged ones (Figure 7D).

**Figure 6.** 2-NBDG uptake and G6PDH activity increase in NO-resistant *L. braziliensis* strain after the nitrosative challenge. Cumulative concentration of all the identified proteins involved in (**A**) glycolysis and (**B**) pentose phosphate pathway (PPP). Each dot represents the total sum of the concentration values of proteins involved in those processes in each of the four biological replicates. Absolute protein concentration of (**C**) phosphotransferase; (**E**) glucose-6-phosphate dehydrogenase (G6PDH); (**F**) transaldolase; and (**G**) 6-phosphofructokinase-1. (**D**) 2-NBDG uptake after parasite incubation for 30 min at 25 ◦C. Median values were obtained by subtraction of fluorescence at 4 ◦C. Graphs represent mean ± SD of at least 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.
