**3.1. NOx at the center of prokaryotic life, virulence, and death**

Several environmental, commensal, and pathogenic prokaryotes possess an arsenal of proteins dedicated to the nitrogen metabolism under anaerobic conditions [67]. Indeed, an enzymatic chain is involved in the denitrification pathway responsible for the production of NO through nitrite reductase (NIR) as an intermediate. The outcome of this mechanism is the formation of dinitrogen (N2 ). This process is crucial for the growth of bacteria in the absence of oxygen because it is coupled with ATP generation (**Figure 12**).

Interestingly, a shortened NOS-like protein called bNOS was discovered in Gram-positive bacteria [28]. This enzyme is deprived of the classical reductase domain already described in other NOS isoforms. However, bNOS is able to recruit other proteins exhibiting a reductase catalytic activity [69]. Similar to mammalian isoforms, bNOS catalyzes the production of NO using l-arginine precursor. However, NOx is key elements in the innate immunity to avoid infection. Indeed, similar to eukaryotic cells, prokaryotes are also sensitive to NOx activity. Thence, such compounds could alter DNA, proteins, and metal centers leading to severe alterations of bacterial growth and replication.

As previously described, NO could be endogenously produced by bacteria but is also a powerful exogenous antimicrobial agent. This highlights the requirement of systems to manage a bearable threshold of NOx within the cell. First, bacteria have developed NO sensing system

**Figure 12.** Bacterial nitrogen cycle (adapted from [68]). NO3 − , nitrate; NO<sup>2</sup> − , nitrite; NO, nitric oxide; N<sup>2</sup> O, nitrous oxide; N2 , dinitrogen; NH<sup>2</sup> OH, hydroxylamine; NH<sup>4</sup> + , ammonia; NAR/NAP, nitrate reductase; NIR, nitrite reductase; NOR, nitric oxide reductase; N<sup>2</sup> OR, nitrous oxide reductase.

through non-heme iron cluster conjugated with a wide range of detoxification mechanisms. Several pathways involving reaction of NO with non-heme iron cluster were described, mainly in *Escherichia coli* [70]. Indeed, NO can lead to a raise of the repression of sulfur assimilation operon (*suf*) exerted by ferric uptake regulator protein (Fur). This reaction is mediated by Fur nitrosylation through NO-derived S-nitrosoglutathione (GSNO). *Suf* products are required for the biogenesis of the [Fe─S] cluster, which, in turn, gives a lure to get rid of NOx collateral damages. The flavohemoglobin (hmpA) is another response against the nitrosative stress. This enzyme catalyzes the conversion of NO into nitrous oxide (N2 O) under anaerobic conditions. Interestingly, in the presence of NO, the S-nitrosylation of cysteine residues on MetR and FNR inactivates both the proteins. Thus, the repression exerted by MetR and the fumarate and nitrate reductase regulatory protein (FNR) is raised by *hmpA* allowing its transcription. Furthermore, NO-responsive transcriptional factor (NorR) is directly activated by NO and enhances the expression of NorVW protein catalyzing the reduction of NO into N<sup>2</sup> O. Redoxsensitive cysteines also take part in the NO-sensing systems. Indeed, NO-activated superoxide regulon (SoxR) protein leads to the transcription increase of superoxide dismutase (*sodA*) removing O2 − that may react with NO to form NO2 and ONOO<sup>−</sup> [71]. Similar to SoxR, peroxide regulon (OxyR) protein is activated by S-nitrosylation. This protein induces gene expression for protective products against nitrosative stress at least by limiting the S-nitrosylation [72]. For example, the expression of catalase KatA, which could buffer free NO, is activated by OxyR [73]. Altogether, these sensing systems lead to the efficient detoxification by generation of less toxic compounds or scavenger molecules.

kinase (HK) or (ii) diguanylate cyclase (DGCs) and phosphodiesterase (PDE). H-NOX could modify the function of proteins implicated in virulence such as biofilm formation and motility or even *quorum sensing* [83]. For example, H-NOX alters the function of diguanylate cyclase and enhances the phosphodiesterase activity. Altogether, this leads to the biofilm dispersion through the decrease of c-di-GMP, an essential mediator in this process. On the contrary, a more sophisticated system relative to histidine kinase (HnoK) activity leads to the accumulation of c-di-GMP in response to NO favoring the biofilm adhesion and formation. Another important virulence trait could be also modulated by H-NOX as in *Vibrio* species a cross-talk

The Hidden Face of Nitrogen Oxides Species: From Toxic Effects to Potential Cure?

http://dx.doi.org/10.5772/intechopen.75822

More recently, an antibiotic resistance modulation was promoted by a bacterial NO2

coupled with an increase in related antibiotic resistance [84]. The link between NO2

exposure. Indeed, an upregulation in expression of MexEF-OprN efflux pump was observed

efflux pump is yet not established, but the authors suggest here a potential role of this protein in the releasing of NOx outside the cells. Previous studies also highlight a correlation between NO endogenously generated by bNOS and increase aminoglycoside resistance [85]. NO and

To resume, bacteria are able to produce NO and RNS. Following endogenous burst or exogenous aggression (pollution, inflammation reactions), a physiological threshold is exceeded. However, bacteria developed an ingenious system to counteract the potential effects of NO and derived products. Various interconnected signaling pathways are implicated in this phenomenon: (i) NO detection, (ii) detoxification, (iii) repairing mechanisms, and (iv) metabolic reprogramming. Finally, these fundamental modifications modulate bacterial community behavior (QS) and virulence traits, such as biofilm and antibiotic resistance. This last point is particularly attractive in medicine. Indeed, the emergence of several antibiotic multiresistant pathogens represents a big threat for human that brings us to the last question of this review:

A lot of therapeutic strategies exploiting NOx-mediated pathways has recently emerged, particularly to treat cancer. Here we propose to classify them in three families: (i) NO-donor compounds, materials, or nanoparticles; (ii) modulators of NOS activity; and (iii) last but not

The first family NO donors consist of NO-chelating compounds that can release NO under specific conditions within the organism after administration. As shown below, several NO donors have been developed during the last decade. Their global—direct or not—anticancer

For further information, we strongly recommend to read the review of Huang et al. [86]. Another extension of the use of NO-donor compounds is their graft on material and nanoparticles. Indeed, prosthetic biomaterials used in medical devices were modified through covalent or noncovalent binding of two NO donors (diazeniumdiolates and S-nitrosothiols).

could directly interact with antibiotic leading to a less toxic compound and exerted a

gaseous

33

and this

with QS seems to exist. However, a few studies are yet available.

repression of bacterial respiration preventing drug uptake.

"Does NOx therapy represent the new trends in human health care?"

effects have been exploited in the case of various carcinoma (**Table 1**).

**3.2. The novel therapeutic strategies mediated by NOx**

least, generators of gaseous NO.

NO2

Moreover, bacteria also develop repairing system of DNA and [Fe─S] cluster damages related to nitrosative stress. Similar to eukaryotic cells, five DNA repair pathways are available in prokaryotes. Among them, the base excision repair (BER), to repair deaminated base, and the nucleotide excision repair (NER), to remove cross-linking in DNA, are predominant [74]. Concerning the [Fe─S] cluster repair, the iron sulfur cluster S protein (IscS), a cysteine desulfurase, can denytrosylate these protein clusters in the presence of l-cysteine [75]. Moreover, Isc regulator (IscR) protein is able to sense nitrosylated [Fe─S] cluster and thus enhances the formation and/or repair of [Fe─S] cluster [76, 77].

NO could also alter tricarboxylic acid (TCA) metabolic pathway and bacterial respiration through reaction with aconitase and cytochromes of the electron transfer chain, respectively [78]. However, these mechanisms are crucial for the generation of ATP and hence the energetic needs of bacteria [79]. To counteract the inefficiency of this enzyme, some bacteria are able to reprogram their metabolism. For example, *Pseudomonas* spp. uses the citrate lyase, phosphoenolpyruvate carboxylase, and pyruvate phosphate dikinase to convert citrate into pyruvate and ATP [80].

In nonlethal concentrations, NO interplays a signaling role in bacteria through several proteins possessing heme-nitric oxide/oxygen-binding domain and is referred under the name H-NOX proteins. H-NOX exhibits a highly conserved domain among bacteria and also shared in mammals (sGC) [81]. All H-NOX are histidine-ligated protoporphyrin IX hemoprotein able to fix NO on its ferrous iron. The H-NOX-encoding genes are found in operons with diverse bacterial signaling genes [82], whose majority is now divided in two classes: containing (i) histidine kinase (HK) or (ii) diguanylate cyclase (DGCs) and phosphodiesterase (PDE). H-NOX could modify the function of proteins implicated in virulence such as biofilm formation and motility or even *quorum sensing* [83]. For example, H-NOX alters the function of diguanylate cyclase and enhances the phosphodiesterase activity. Altogether, this leads to the biofilm dispersion through the decrease of c-di-GMP, an essential mediator in this process. On the contrary, a more sophisticated system relative to histidine kinase (HnoK) activity leads to the accumulation of c-di-GMP in response to NO favoring the biofilm adhesion and formation. Another important virulence trait could be also modulated by H-NOX as in *Vibrio* species a cross-talk with QS seems to exist. However, a few studies are yet available.

More recently, an antibiotic resistance modulation was promoted by a bacterial NO2 gaseous exposure. Indeed, an upregulation in expression of MexEF-OprN efflux pump was observed coupled with an increase in related antibiotic resistance [84]. The link between NO2 and this efflux pump is yet not established, but the authors suggest here a potential role of this protein in the releasing of NOx outside the cells. Previous studies also highlight a correlation between NO endogenously generated by bNOS and increase aminoglycoside resistance [85]. NO and NO2 could directly interact with antibiotic leading to a less toxic compound and exerted a repression of bacterial respiration preventing drug uptake.

To resume, bacteria are able to produce NO and RNS. Following endogenous burst or exogenous aggression (pollution, inflammation reactions), a physiological threshold is exceeded. However, bacteria developed an ingenious system to counteract the potential effects of NO and derived products. Various interconnected signaling pathways are implicated in this phenomenon: (i) NO detection, (ii) detoxification, (iii) repairing mechanisms, and (iv) metabolic reprogramming. Finally, these fundamental modifications modulate bacterial community behavior (QS) and virulence traits, such as biofilm and antibiotic resistance. This last point is particularly attractive in medicine. Indeed, the emergence of several antibiotic multiresistant pathogens represents a big threat for human that brings us to the last question of this review: "Does NOx therapy represent the new trends in human health care?"
