**2. Formation of reactive oxygen and nitrogen species by activated macrophages**

Upon phagocytosis, NADPH oxidase (NADPHox) assembles into an enzymatically active complex that transfers electrons from NADPH to molecular oxygen producing superoxide

Thiol-Dependent Peroxidases in *Mycobacterium tuberculosis* Antioxidant Defense 295

oxidizing and nitrating moiety (Ferrer-Sueta & Radi, 2009; Alvarez *et al.*, 2011). In the absence of direct targets, peroxynitrous acid (p*K*a = 6.5-6.8, (Goldstein & Czapski, 1995; Pryor & Squadrito, 1995; Kissner *et al.*, 1997)) homolyses into nitrogen dioxide (•NO2) and •OH in 30% yields (*k* = 0.9 s-1 at pH 7.4 and 37 ºC) (Goldstein & Czapski, 1995; Gerasimov & Lymar, 1999). However, the importance of this reaction *in vivo* is probably limited, since in cells, most peroxynitrite is expected to be involved in direct reactions. For instance, peroxynitrite can react with carbon dioxide (CO2) present in mM concentrations in biological systems (*k* = 4.6 x 104 M-1 s-1 at pH 7.4 and 37 ºC), leading to the formation of up to 35% carbonate (CO3•-) and •NO2 radicals, which are also oxidizing species (Lymar & Hurst, 1995; Denicola *et al.*, 1996; Bonini *et al.*, 1999; Augusto *et al.*, 2002). •OH and •NO2 can participate in lipid peroxidation reactions, resulting in fatty acid hydroperoxide formation (Barber & Thomas, 1978; Prutz *et al.*, 1985). These can also be synthesized by enzymatic mechanisms through lipoxygenase (LOX)-catalyzed reactions (Sevanian *et al.*, 1983). Fatty acid hydroperoxides can be released from membranes by the action of phospholipase A2 (PLA2)(Bonney *et al.*, 1985). Free arachidonic acid is toxic for *M. tuberculosis* acting in a synergistic way with reactive nitrogen species (Akaki *et al.*, 2000). Although the mechanism of synergism has not been resolved, the fact that free fatty acid- dependent toxicity to *Helicobacter pylori* increases in peroxidase-deficient strains indicates that fatty acid hydroperoxides could participate in cytotoxicity (Wang *et al.*, 2006). In summary, inside the phagosomes of activated macrophages, and among other reactive species, different peroxides can be formed, including H2O2, peroxynitrite and fatty acid hydroperoxides (Figure 1). All of these species have been reported to be cytotoxic against microorganisms including bacteria (Clifford & Repine, 1982; Denicola *et al.*, 1993; Hurst & Lymar, 1997; Evans *et al.*, 1998; Wang *et al.*, 2006). The enzymatic mechanisms that allow reactive nitrogen and oxygen species detoxification, in general, and peroxide reduction, in particular, thus enabling the bacterium to infect and persist inside the phagosome of activated macrophages,

**3. Singular aspects of the antioxidant defense systems of** *M. tuberculosis*

The antioxidant defenses of *Mycobacterium tuberculosis* are unusual in many aspects. As most other Actinobacteria, it lacks glutathione, and contains millimolar concentration of 1-D-myo-inosityl-2-deoxy-2-(N-acetyl-L-cysteinyl)amino-D-glucopyranoside, or mycothiol (MSH), as main low molecular weight thiol (Newton & Fahey, 2002). MSH is maintained in the reduced form by mycothione reductase using NADPH as electron donor (Patel & Blanchard, 2001). It participates in drug detoxification pathways by forming adducts with alkylating agents and antibiotics that are subsequently cleaved by MSH S-conjugate amidase to generate a mercapturic acid (excreted outside the cell) and glucosamine inositol (used to regenerate MSH) (Newton *et al.*, 2000). MSH can function as a resource for metabolic precursors and for energy production (Bzymek *et al.*, 2007). Mycothiol-deficient *M. smegmatis* strains are more sensitive to •NO- and H2O2-mediated toxicity than wild type strains (Rawat *et al.*, 2002; Miller *et al.*, 2007). However, there is currently no evidence for MSH acting as a reducing substrate for any peroxidase. Mycobacteria, among other organisms, also synthesize ergothioneine, which is a thiourea derivative of histidine containing a sulfur atom in the imidazole ring. Its synthesis is increased in *M. smegmatis* mutants in MSH synthesis suggesting a compensation mechanism (Ta *et al.*, 2011), although the actual function of this unusual thiol remains to

is a field of active investigation.

anion radical (O2•-) inside the phagosomes (Babior, 1984; Groemping & Rittinger, 2005). The charged nature of this radical at physiological pH (hydroperoxyl radical (HO2•) p*K*a = 4.75, (Blelski & and Allen, 1977)) determines its low diffusion capability through membranes. In turn, INFγ-mediated induction of iNOS leads to the formation of nitric oxide (•NO), a small lipophilic moiety that can diffuse into the phagosome (Xie *et al.*, 1993; Martin *et al.*, 1994; MacMicking *et al.*, 1997). O2 •- can spontaneously or enzymatically dismutate into hydrogen peroxide (H2O2)(De Groote *et al.*, 1997; Fridovich, 1997). Reactions of the latter species with reduced transition metal centers (particularly containing Fe2+ or Cu+) yield the strong and non-selective oxidizing compound, hydroxyl radical (•OH) through Fenton reactions. Moreover, the diffusion-controlled reaction between O2 •- and •NO forms peroxynitrite1, an

Fig. 1. **Peroxide sources in** *M. tuberculosis* **(***Mt***)-harboring phagosomes of activated macrophages.** Details on the pathways leading to the production of peroxides (H2O2, peroxynitrite and fatty acid hydroperoxides (FA-OOH), in bold) among other reactive nitrogen and oxygen species are given in the text. Dashed lines indicate reactions involving several steps and intermediates.

<sup>1</sup> IUPAC recommended names for peroxynitrite anion (ONOO- ) and peroxynitrous acid (ONOOH) are oxoperoxonitrate (1-) and hydrogen oxoperoxonitrate, respectively. The term peroxynitrite is used to refer to the sum of ONOO and ONOOH.

anion radical (O2•-) inside the phagosomes (Babior, 1984; Groemping & Rittinger, 2005). The charged nature of this radical at physiological pH (hydroperoxyl radical (HO2•) p*K*a = 4.75, (Blelski & and Allen, 1977)) determines its low diffusion capability through membranes. In turn, INFγ-mediated induction of iNOS leads to the formation of nitric oxide (•NO), a small lipophilic moiety that can diffuse into the phagosome (Xie *et al.*, 1993; Martin *et al.*, 1994;

peroxide (H2O2)(De Groote *et al.*, 1997; Fridovich, 1997). Reactions of the latter species with reduced transition metal centers (particularly containing Fe2+ or Cu+) yield the strong and non-selective oxidizing compound, hydroxyl radical (•OH) through Fenton reactions.

Fig. 1. **Peroxide sources in** *M. tuberculosis* **(***Mt***)-harboring phagosomes of activated macrophages.** Details on the pathways leading to the production of peroxides (H2O2, peroxynitrite and fatty acid hydroperoxides (FA-OOH), in bold) among other reactive nitrogen and oxygen species are given in the text. Dashed lines indicate reactions involving

oxoperoxonitrate (1-) and hydrogen oxoperoxonitrate, respectively. The term peroxynitrite is used to

•- can spontaneously or enzymatically dismutate into hydrogen

•- and •NO forms peroxynitrite1, an

) and peroxynitrous acid (ONOOH) are

MacMicking *et al.*, 1997). O2

several steps and intermediates.

refer to the sum of ONOO-

1 IUPAC recommended names for peroxynitrite anion (ONOO-

and ONOOH.

Moreover, the diffusion-controlled reaction between O2

oxidizing and nitrating moiety (Ferrer-Sueta & Radi, 2009; Alvarez *et al.*, 2011). In the absence of direct targets, peroxynitrous acid (p*K*a = 6.5-6.8, (Goldstein & Czapski, 1995; Pryor & Squadrito, 1995; Kissner *et al.*, 1997)) homolyses into nitrogen dioxide (•NO2) and •OH in 30% yields (*k* = 0.9 s-1 at pH 7.4 and 37 ºC) (Goldstein & Czapski, 1995; Gerasimov & Lymar, 1999). However, the importance of this reaction *in vivo* is probably limited, since in cells, most peroxynitrite is expected to be involved in direct reactions. For instance, peroxynitrite can react with carbon dioxide (CO2) present in mM concentrations in biological systems (*k* = 4.6 x 104 M-1 s-1 at pH 7.4 and 37 ºC), leading to the formation of up to 35% carbonate (CO3•-) and •NO2 radicals, which are also oxidizing species (Lymar & Hurst, 1995; Denicola *et al.*, 1996; Bonini *et al.*, 1999; Augusto *et al.*, 2002). •OH and •NO2 can participate in lipid peroxidation reactions, resulting in fatty acid hydroperoxide formation (Barber & Thomas, 1978; Prutz *et al.*, 1985). These can also be synthesized by enzymatic mechanisms through lipoxygenase (LOX)-catalyzed reactions (Sevanian *et al.*, 1983). Fatty acid hydroperoxides can be released from membranes by the action of phospholipase A2 (PLA2)(Bonney *et al.*, 1985). Free arachidonic acid is toxic for *M. tuberculosis* acting in a synergistic way with reactive nitrogen species (Akaki *et al.*, 2000). Although the mechanism of synergism has not been resolved, the fact that free fatty acid- dependent toxicity to *Helicobacter pylori* increases in peroxidase-deficient strains indicates that fatty acid hydroperoxides could participate in cytotoxicity (Wang *et al.*, 2006). In summary, inside the phagosomes of activated macrophages, and among other reactive species, different peroxides can be formed, including H2O2, peroxynitrite and fatty acid hydroperoxides (Figure 1). All of these species have been reported to be cytotoxic against microorganisms including bacteria (Clifford & Repine, 1982; Denicola *et al.*, 1993; Hurst & Lymar, 1997; Evans *et al.*, 1998; Wang *et al.*, 2006). The enzymatic mechanisms that allow reactive nitrogen and oxygen species detoxification, in general, and peroxide reduction, in particular, thus enabling the bacterium to infect and persist inside the phagosome of activated macrophages, is a field of active investigation.
