*3.8. Nitrosative Challenge Impairs Mitochondrial O2 Consumption by L. braziliensis Strains*

Mitochondrial respiration and, specifically, proteins involved in oxidative phosphorylation (OXPHOS) are inhibited by NO due to competition with oxygen [41]. Here, we observed that oxygen consumption in routine condition is 61.5% lower in 2856 strain than in 2853. Additionally, the mitochondrial respiration is more affected by NaNO2 treatment in NO-susceptible parasites than in NO-resistant. Thus, although nitrosative stress induced a significant decrease in both strains, NO-resistant parasites can maintain oxygen consumption 1.7-fold higher than NO-susceptible (Figure 8). Interestingly, ROX state, which indicates mitochondrial-independent oxygen consumption, increased~3.5-fold in 2856 strain when compared to 2853, suggesting higher ROS production by NO-susceptible parasites. Besides that, the NO exposure decreased the oxygen consumption to similar levels during ROX state in both strains (Figure 8).

**Figure 7.** Nitrosative challenge increases D-lactate dehydrogenase (D-LDH) abundance in NOresistant *L. braziliensis* strain. (**A**) Protein concentration of pyruvate kinase; (**B**) cumulative concentration of 6-phosphofructo-2-kinase; (**C**) protein concentration of D-lactate dehydrogenase (D-LDH); and (**D**) LDH activity in parasite homogenates. Graphs represent mean ± SD of at least four independent experiments. Statistical differences by Student's *t* test (\* *p* < 0.05; \*\* *p* < 0.01). 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.

Based on these results, the abundance of mitochondrial complexes, as well as the concentration levels of several proteins, were analyzed. First, we observed that there were no differences in the concentration of citrate synthase, an enzyme associated with mitochondrial integrity, among the experimental groups (Figure 9A). Notably, we observed a significant decrease in the accumulated abundance of the proteins involved in OXPHOS in the NO-resistant parasites after the NO challenge (Figure 9B). Intriguingly, the protein concentration of complexes I, II, and IV is significantly higher in NO-susceptible parasites than in NO-resistant, and the exposure to nitrosative stress does not affect the abundance of these proteins (Figure 9C,D,F). In contrast, protein concentration of complex V is significantly lower in NO-susceptible parasites than in NO-resistant ones (Figure 9G). Interestingly, upon nitrosative challenge, parasites of 2853 strain increase the concentration of molecules related to complex I and decrease the abundance of complexes III and V (Figure 9C,E,G). Ubiquinone acts as an electron carrier from complexes I and II to complex III. Interestingly, we found that the concentration of a protein that participates in the biosynthesis of this molecule is significantly decreased in NO-susceptible parasites during nitrosative condition (Figure 9H), suggesting that 2856 strain may be unable to maintain the ubiquinone pool under NO exposure.

**Figure 8.** Nitrosative challenge impairs mitochondrial O2 consumption by *L. braziliensis* strains. Parasites were incubated in the respiration buffer at 25 ◦C to evaluate the O2 uptake under routine condition and after the addition of 2 μM AA (ROX state). Graphs represent mean ± SD of four independent experiments. Significance of differences between treatments were determined by two-way ANOVA followed by Tukey's multiple comparisons test (\* *p* < 0.05; \*\* *p* < 0.001; and \*\*\*\* *p* < 0.0001). 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.

**Figure 9.** Proteins of mitochondrial oxidative phosphorylation (OXPHOS) are differentially modulated in *L. braziliensis* strains. (**A**) Cumulative concentration of all the identified proteins as citrate synthase. Cumulative concentration of all the identified proteins involved in (**B**) OXPHOS; (**C**) NADHubiquinone oxidoreductase (complex I); (**D**) succinate ubiquinone oxidoreductase (complex II); (**E**) ubiquinol:cytochrome c oxidoreductase (complex III); (**F**) cytochrome c oxidase (complex IV); and (**G**) FoF1-ATP synthase (complex V). Each dot represents the total sum of the concentration values

of proteins involved in those processes or complexes in each of the four biological replicates. (**H**) Absolute protein concentration of ubiquinone biosynthesis protein. Graphs represent mean ± SD of at least four independent experiments. Statistical differences by Student's *t* test (\* *p* < 0.05; \*\* *p* < 0.01; and \*\*\* *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.

## **4. Discussion**

To complete its life cycle, *Leishmania* must overcome several barriers found in the vertebrate host, such as increased temperature (25 ◦C in insect vector to 37 ◦C in mammalian host), pH acidification in phagolysosome, and changes of available carbon sources [42]. Moreover, the parasite must survive the oxidative burst and NO production, two main microbicidal mediators that started after macrophage activation [43]. Although the host immune response is directly related to the prognosis of leishmaniasis [44], intrinsic virulence features of *Leishmania* strains are also decisive for infection and clinical outcome [21]. In the present study, we evaluated two strains of *L. braziliensis* with polarized phenotype of susceptibility or resistance to NO, which were also associated with responsiveness or refractoriness to antimony treatment [17,20]. Aspects related to nitrosative and oxidative stresses suffered by the parasites during host cells infection, in addition to a deep analysis of parasites' proteome-mediated mechanisms for resisting to NO were considered. It is important to point out that during the very initial steps of infection, promastigotes are subjected to the early oxidative burst triggered into the innate immunity cells -at the same time host and effectors- in response to the invasion. To cope with this, promastigotes should display their repertoire of antioxidant responses. Depending on the success during such very early responses, parasites will survive, and differentiate, determining the successful parasite colonization and further persistence. Our proteomics and biochemical data on promastigotes challenged with NaNO2 would reflect these very initial parasite responses.

First, in vitro infection allowed for the identification of differences between both strains, corroborating the higher infection capability of NO-resistant parasites, both in terms of number of infected macrophages, as well as in number of amastigotes per cell. Our findings were similar to those of Souza et al. [20], who used human macrophages for in vitro evaluation of *L. braziliensis* infection. Interestingly, we also observed that both strains trigger host microbicidal responses through increased production of ROS and RNS, with levels exacerbated in the infection produced by NO-resistant strain. In cutaneous leishmaniasis, the development of a non-healing course of infection has been linked to an immunosuppressive profile, with increased arginase 1 activity [45], a cytosolic enzyme that favors parasites' antioxidant metabolism and that, at the same time, competes with iNOS for the common substrate L-arginine, decreasing NO production [46,47]. Although Costa et al. [21] demonstrated that this process also occurs during in vivo infection of BALB/c mice with the NO-resistant *L. braziliensis* strain, it is only elicited in late infection times (seven weeks post-infection). All together, these data suggest that NO-resistant parasites are endowed with specific mechanisms to evade host defenses, raising a question about the phenotypic adaptations that allow this strain to survive in the presence of high concentrations of toxic molecules, especially during early infection course.

According to previous studies, *L. braziliensis* amastigotes survive and replicate much better in the absence of ROS, identifying these molecules as important regulators of the parasite proliferation inside the host cells [48]. In addition, supplementation of cell cultures with pro-oxidants and antioxidants modulates *Leishmania* infection, resulting in a reduced parasite load in stressful conditions [49]. Moreover, sensitivity to distinct reactive species may vary in the parasite stages, with H2O2 being more toxic to *Leishmania* amastigotes than O2 •− [43,50]. In line with those observations, infection with the NO-susceptible strain reproduces all phenotypes previously described, including the high sensitivity to H2O2 (comparing the infection index of catalase- and SOD-treated cells). In contrast, the infection with NO-resistant parasites has unique features; only increased nitrosative stress derived

from NaNO2-treated macrophages was able to downregulate the infection by this strain. Resistant parasites also appear to be unaffected by H2O2 since neither this reactive species (even with the increase in RNS production) nor the presence of catalase were able to change the infection index. In contrast, O2 •− metabolism might be essential for NO-resistant strain because only SOD-treated cells showed reduction in the parasite load. The use of tempol, an antioxidant able to promote O2 •− dismutation at rates similar to SOD, pointed to the dual role of this free radical to control the infection, highlighting differences in the pathogenesis of cutaneous leishmaniasis caused by different species of *Leishmania* [49,51,52].

To shed light on the molecular mechanisms underlying the NO-resistant phenotype in *L. braziliensis*, we performed an unbiased and comprehensive quantitative analysis of parasites' proteome. Notably, differences in total protein per cell and absolute protein concentrations clearly distinguished NO-resistant from NO-susceptible parasites, corroborating the notion that there are natural intrinsic differences at proteome level among *L. braziliensis* strains circulating in a same geographical region [9]. In addition, we demonstrated that the rapid modulation of NO-resistant parasites' proteome upon NO challenge involves an increase in total protein content, which is suggestive of ploidy alterations in response to nitrosative stress. In line with this proposal, recently it was reported that aneuploidy in *L. donovani* is followed by proteome modulation and could explain metabolic differences between strains [53]. Aneuploidy and karyotypic mosaicism are common in *Leishmania* spp., and such genome plasticity allow parasites to explore fitness possibilities for survival [54–56]. In *Leishmania*, aneuploidy is a species- and strain-specific trait that varies according to external stimuli, including drug pressure, enabling rapid adaptation to hostile conditions [57–60]. However, aneuploidy may also result in metabolic alterations that lead to oxidative and proteotoxic stresses [61]. Interestingly, we observed that in contrast to NO-resistant strain, the NO-susceptible parasites naturally (without NO stimulus) present a protein content higher than expected and are not able to modulate it upon NO challenge, suggesting that NO-susceptible parasites are "naturally stressed" and that proteome (and genome) plasticity in these parasites probably reached a limit. Remarkably, the deep proteomics approach conducted in this study allows one to clearly differentiate resistant parasites from susceptible ones and reveals the subset of proteins that explain those phenotypes.

Proteomic profiling of *L. donovani* parasites adapted to sub-lethal doses of NO donor showed the upregulation of several parasite's proteins involved in the ROS detoxification pathway, suggesting a cross-resistance to both nitrosative and oxidative stresses [62]. Accordingly, we observed a positive modulation in the concentration of proteins associated with *"cell redox homeostasis and response to oxidative stress*" in NO-resistant parasites, even in the condition without the NO donor. In addition, the challenge with NaNO2 specifically affected the concentration of APx, increasing protein abundance in NO-resistant parasites and decreasing in NO-susceptible ones. Sardar et al. [61] also demonstrated that exposure of *L. donovani* promastigotes to ROS or RNS elevates APx protein abundance up to 2.5-fold, whereas combination of both stresses produced an additive effect, increasing this protein in 3.2-fold. Additionally, ROS-inducible APx of *L. amazonensis* is essential for parasite infectivity, replication, and virulence in vitro and in vivo models [63]. Collectively, these data support the idea that NO-resistance in *L. braziliensis* is associated with APx increased abundance as a response to stressful conditions, which also explains the successful intracellular replication of NO-resistant parasites in macrophages overproducing H2O2. Interestingly, *L. braziliensis* APx-overexpressing parasites have an 8-fold increase in the antimony-resistance index [64]. As *L. braziliensis* NO-resistant parasites used here are also refractory to antimony treatment, our data reinforce the idea that APx could participate in both NO- and Sb-resistance.

Regarding the T(SH)2/TR system, we observed a decrease in TR abundance after NO challenge. Although this phenomenon occurred to a lesser extent in NO-resistant parasites, such reduction indicates that other mechanisms are concurring to maintain the pool of reducing intermediates. Despite the fact that T(SH)2 is a much more efficient scavenger

of hydroperoxides than other thiols present in trypanosomatids [65], all these protozoa possess glutathione-dependent enzymes [66]. Susceptibility of different *Leishmania* species to the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) was inversely correlated with the levels of GSH but not with their total thiol content (including T(SH)2 levels) [67]. In addition, *L. donovani* showed ~2.9-fold upregulation of glutathione peroxidase-like protein in detrimental to TR in response to nitrosative stress [62]. In agreement with those observations, our results show that *L. braziliensis* NO-resistant parasites have higher concentrations of proteins involved in GSH biosynthesis pathway, particularly GSH1, and GPx activity than NO-susceptible parasites upon NO exposure. Such increased levels can avoid the toxic effects of nitrosative stress and oxidative burst found after treatment with NO donor, or during host infection. Additionally, we cannot rule out the role of GSH pool in regeneration of ascorbate, a cofactor of APx, since in several organisms this is the main ascorbate recycling system [68]. Although in trypanosomatids this process seems to be achieved via T(SH)2 [69], further analyses should be performed to evaluate the activity of GSH in *L. braziliensis* parasites, especially in NO-resistant ones.

In *L. donovani,* the response to oxidative stress requires a rapid metabolic reconfiguration of glucose metabolism, involving a shift from glycolysis toward PPP for replenishment of NADPH pool when parasites are exposed to oxidants. mRNA and protein levels of PPP enzymes of the oxidative and nonoxidative branch, such as G6PDH and transaldolase, were up-regulated in promastigotes exposed to sublethal doses of pro-oxidants, while the viability of promastigotes treated with G6PD inhibitor and sublethal doses of ROS was restored by coincubation with N-acetyl cysteine or GSH [70]. In addition, cell lines overexpressing G6PDH and transaldolase are also more resistant to antimonial, amphotericin B, and miltefosine [70]. In agreement, our dataset indicates that NO resistance in *L. braziliensis* also involves a rapid shift in glucose metabolism from glycolysis to PPP in response to nitrosative stress, without the detriment of glycolysis per se. Protein concentration of G6PDH and transaldolase were increased in NO-resistant parasites and decreased in NOsusceptible ones in response to the NO challenge. Such increase in protein concentration enables NO-resistant parasites to replenish intracellular NADPH levels and, consequently, the GSH pool, to maintain its cellular redox balance. The proteome of promastigotes of *L. infantum* strains resistant to NO also showed increased abundance of G6PDH, while amastigotes of *L. infantum* cell lines selected for NO-resistant exhibited overexpression of 6-phosphogluconate dehydrogenase mRNA levels [23,71], reinforcing the notion that the PPP is an indispensable pathway for resistance to nitrosative stress.

Intriguingly, we observed a significant increase in the concentration of D-LDH in NOresistant parasites after exposure to NO, and such increase was accompanied by increased LDH enzymatic activity. In *L. major,* D-lactate produced via methylglyoxal metabolism would be converted to pyruvate by a D-LDH, with the resulting molecule feeding the tricarboxylic acid cycle (TCA) and other pathways [72–74]. As the methylglyoxal is a toxic glycolytic metabolite, we hypothesize that the increase in glucose uptake and, consequently, in the production of methylglyoxal may occur in response to nitrosative stress in NOresistant parasites challenged with NO, leading to upregulation of D-LDH abundance and activity in these parasites. However, further assays need to be done to demonstrate the accumulation of that metabolite in parasites under nitrosative stress. Interestingly, and supporting once again the idea that NO- and drug-resistance are related, recently it was observed that transcript levels coding for D-LDH increased 56-fold in a *L. donovani* cell line selected for paromomycin resistance and that wild-type parasites transfected with *D-LDH* acquired a significant resistance against the drug [75].

It has been suggested that a metabolic shift from glycolysis to mitochondrial respiration is detrimental for *L. mexicana* virulence in vivo, leading to high ROS production and increased sensitivity of parasites to NO. Therefore, the maintenance of glucose uptake would be an advantage in the oxidative environment of the phagolysosome [76]. Accordingly, in NO-resistant parasites, apart the complex I, the concentration of all components of mitochondrial electron transport system was not modified or was even diminished (complexes

III and V) after NO challenge. Such results may explain the drastic decrease in oxygen consumption by NO-resistant strain upon NO exposure. This scenario is extremely interesting and raises several possibilities about the metabolic adaptations suffered by *L. braziliensis*. For an efficient microbicidal response, macrophages need to activate both NADPH oxidases and iNOS, which can cause hypoxia conditions and lead to expression of hypoxia-inducible factor-1α (HIF-1α) by host cells [45]. In patients with ATL caused by *L. braziliensis,* the expression of this transcription factor has already been described, suggesting hypoxia participation during cutaneous and mucocutaneous clinical outcomes [77]. As described by Degrossoli et al. [78], low oxygen tension, derived from enhanced ROS generation, also leads to the reduction of intracellular parasites in *L. amazonensis*-infected macrophages. Together, these data may suggest that the phagolysosomal environment, rich in NO and ROS, is not an attractive place to perform OXPHOS and derived metabolisms since they are dependent on oxygen availability. A model reconstruction of energy metabolism in *L. infantum* suggested a reduction in oxygen intake in the amastigote scenario, in comparison to promastigotes, probably indicating the adaptation of amastigote metabolism to hypoxic environment of the macrophage [79]. Hence, the observed increase in glucose uptake and the decrease in mitochondrial oxygen consumption after NO challenge could reflect the adaptations that amastigote forms of NO-resistant *L. braziliensis* should undergo seek to survive in host cells. In addition, complex I would be increased to maintenance of NADH/NAD+ ratio since complex I seems to conserve all subunits containing the redox centers required to ubiquinone reduction [80,81].
