**Nitric Oxide Resistance in** *Leishmania* **(***Viannia***)** *braziliensis* **Involves Regulation of Glucose Consumption, Glutathione Metabolism and Abundance of Pentose Phosphate Pathway Enzymes**

**Nathalia Pinho 1,†, Ana Cristina Bombaça 2,†, Jacek R. Wi´sniewski 3, Geovane Dias-Lopes <sup>4</sup> , Leonardo Saboia-Vahia 1,‡, Elisa Cupolillo <sup>1</sup> , José Batista de Jesus 5, Roque P. de Almeida 6, Gabriel Padrón 1,§ , Rubem Menna-Barreto 2,\* and Patricia Cuervo 1,\***

	- ‡ Current address: Laboratório de Virus Respiratórios e Sarampo, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro 21040-360, RJ, Brazil.
	- § Current address: Center for Genetic Engineering & Biotechnology, La Habana 10600, Cuba.

**Abstract:** In American Tegumentary Leishmaniasis production of cytokines, reactive oxygen species and nitric oxide (NO) by host macrophages normally lead to parasite death. However, some *Leishmania braziliensis* strains exhibit natural NO resistance. NO-resistant strains cause more lesions and are frequently more resistant to antimonial treatment than NO-susceptible ones, suggesting that NO-resistant parasites are endowed with specific mechanisms of survival and persistence. To tests this, we analyzed the effect of pro- and antioxidant molecules on the infectivity in vitro of *L. braziliensis* strains exhibiting polar phenotypes of resistance or susceptibility to NO. In addition, we conducted a comprehensive quantitative mass spectrometry-based proteomics analysis of those parasites. NO-resistant parasites were more infective to peritoneal macrophages, even in the presence of high levels of reactive species. Principal component analysis of protein concentration values clearly differentiated NO-resistant from NO-susceptible parasites, suggesting that there are natural intrinsic differences at molecular level among those strains. Upon NO exposure, NO-resistant parasites rapidly modulated their proteome, increasing their total protein content and glutathione (GSH) metabolism. Furthermore, NO-resistant parasites showed increased glucose analogue uptake, and increased abundance of phosphotransferase and G6PDH after nitrosative challenge, which can contribute to NADPH pool maintenance and fuel the reducing conditions for the recovery of GSH upon NO exposure. Thus, increased glucose consumption and GSH-mediated redox capability may explain the natural resistance of *L. braziliensis* against NO.

**Citation:** Pinho, N.; Bombaça, A.C.; Wi´sniewski, J.R.; Dias-Lopes, G.; Saboia-Vahia, L.; Cupolillo, E.; de Jesus, J.B.; de Almeida, R.P.; Padrón, G.; Menna-Barreto, R.; et al. Nitric Oxide Resistance in *Leishmania* (*Viannia*) *braziliensis* Involves Regulation of Glucose Consumption, Glutathione Metabolism and Abundance of Pentose Phosphate Pathway Enzymes. *Antioxidants* **2022**, *11*, 277. https://doi.org/10.3390/ antiox11020277

Academic Editor: Serge Ankri

Received: 30 December 2021 Accepted: 23 January 2022 Published: 29 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** *Leishmania braziliensis*; nitric oxide resistance; American Tegumentary Leishmaniasis (ATL); nitrosative stress; reactive oxygen species; reactive nitrogen species; quantitative proteomics; FASP; mass spectrometry; glycolysis; glutathione metabolism

#### **1. Introduction**

*Leishmania* (*Viannia*) *braziliensis*, a New World Leishmania species, is an important etiological agent of American Tegumentary Leishmaniasis (ATL) in the Americas [1]. Infection with this species may have clinical outcomes, ranging from self-healing localized cutaneous lesions (LCL) to severe disseminated cutaneous and mucocutaneous forms that may result in facial mutilation by destruction of the palate and nose cartilage. Furthermore, disseminated cutaneous leishmaniasis (DL) and mucocutaneous leishmaniasis (MCL) caused by *L. braziliensis* are frequently refractory to treatment and result from the metastatic dissemination of the parasite [2]. Clinical manifestations result from both the host immune responses and the infecting parasite [3–5]. Indeed, intrinsic characteristics of parasites enable the subversion and/or immunomodulation of host immune responses, resulting in the inactivation of cell pathways crucial for parasite elimination [6–9].

Successful establishment of infection and further parasite persistence depend on a complex interaction between the *Leishmania*'s immune subversion arsenal and the microbicidal mechanisms of mononuclear phagocytes [10]. Such mechanisms include lysosomal enzymes, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Oxidative burst and release of nitric oxide (NO) are the most effective mechanisms against *Leishmania* spp. [11,12]. ROS and RNS can be released when the macrophages are stimulated by TNF-α and IFN-γ, and their production is induced by the activation of routes that involve NADPH oxidases and inducible nitric oxide synthase (iNOS). When it happens, parasites are exposed to superoxide anion (O2 •−), hydrogen peroxide (H2O2), peroxynitrite (ONOO−) and NO [13], which usually results in parasites elimination [11,14,15]. However, some *Leishmania* parasites present natural resistance to NO, helping them to escape the macrophage microbicidal responses and promoting their survival and persistence [16–19]. Indeed, *L. braziliensis* strains isolated from patients with different clinical forms of ATL exhibit different levels of natural resistance to NO. In vitro assays showed that amastigotes from NO-resistant strains survived and multiplied more in human macrophages. In addition, patients infected with NO-resistant parasites presented significantly more severe cutaneous lesions than those infected with NO-susceptible parasites [17].

In vitro infections of human macrophages with *L. braziliensis* NO-resistant strains showed a higher percentage of infected cells and reduced levels of TNF-α production than infections with NO-sensitive parasites. Furthermore, these NO-resistant strains were also correlated with higher refractoriness to pentavalent antimony, the first-line treatment for ATL, suggesting that NO resistance may be related to antimony resistance [20]. In addition, BALB/c mice infected with NO-resistant strains produce more IL-4, which stimulates increased expression of arginase-1, favoring parasites survival and severe forms of disease [21]. Additionally, *L. infantum* field isolates from relapse cases of visceral leishmaniasis are more resistant to antimonial and NO, as well as are more infective to macrophages in vitro, than parasites isolated from responsive patients [22]. However, the mechanisms by which *L. braziliensis* strains can resist/evade the nitrosative stress imposed by host cells have not been clearly defined. Potential mechanisms might include increased abundance of glucose-6-phosphate dehydrogenase (G6PDH), as shown in the study by 2DE-MS of the proteome of *L. infantum* strains resistant or susceptible to NO [23]. Nevertheless, an in-depth study of NO resistance in *Leishmania* is missing.

Based on the previous evidence, we hypothesize that the proteome of *L. braziliensis* NO-resistant strains is tailored to deal with nitrosative stress and, that upon NO challenge, it can be rapidly regulated to minimize the damages caused by nitric oxide. Such adaptations grant the survival and persistence of parasites, leading to chronic infections that

are refractory to treatment. To test this, we performed an unbiased and comprehensive quantitative proteomic analysis of NO-resistant and NO-susceptible *L. braziliensis* strains. By using previously reported procedures for parasite sample preparation and absolute label-free protein quantification [24,25], we were able to compare those parasites before and after stimulus with an NO donor, identifying ~6300 proteins and estimating absolute concentrations of ~6000 of these proteins. We also evaluated the effect of nitrosative and oxidative stresses, as well as the effect of antioxidant molecules, on the infection index of each strain. Together, our data provide new evidence on the potential mechanisms underlying the NO resistance in *L. braziliensis*, including increased antioxidant capability involving the glutathione (GSH) system and rapid regulation of glycolysis and pentose phosphate pathway (PPP). Data are available via ProteomeXchange with identifier PXD029462.
