Hydrogen Sulfide in Physiology and Pathogenesis of Bacteria and Viruses

Summary An increasing number of studies have established hydrogen sulfide (H2S) gas as a major cytoprotectant and redox modulator. Following its discovery, H2S has been found to have pleiotropic effects on physiology and human health. H2S acts as a gasotransmitter and exerts its influence on gastrointestinal, neuronal, cardiovascular, respiratory, renal, and hepatic systems. Recent discoveries have clearly indicated the importance of H2S in regulating vasorelaxation, angiogenesis, apoptosis, ageing, and metabolism. Contrary to studies in higher organisms, the role of H2S in the pathophysiology of infectious agents such as bacteria and viruses has been less studied. Bacterial and viral infections are often accompanied by changes in the redox physiology of both the host and the pathogen. Emerging studies indicate that bacterial-derived H2S constitutes a defense system against antibiotics and oxidative stress. The H2S signaling pathway also seems to interfere with redox-based events affected on infection with viruses. This review aims to summarize recent advances on the emerging role of H2S gas in the bacterial physiology and viral infections. Such studies have opened up new research avenues exploiting H2S as a potential therapeutic intervention.


Introduction
Early life forms first appeared on an anoxic earth in the Archean eon, approximately 3.8 billion years ago (1,2). Among them, were the dissimilatory sulfate-reducing bacteria which Amit Singh: http://orcid.org/0000-0001-6761-1664 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. constitute one of the oldest forms of bacterial life on earth. These bacteria utilized inorganic sulfur substrates and produced hydrogen sulfide (H 2 S) as the end product of anaerobic respiration (3). Before the "great oxidation event" which occurred 2.5 billion years ago leading to an increase in atmospheric oxygen, H 2 S remained the most abundant and versatile chemical on the primitive earth (4,5). In fact, H 2 S is widely believed to be the primordial sustainable energy source (6). Primitive photolithotrophs used sulfide as the terminal electron acceptor, similar to today's green and purple sulfur bacteria. Therefore, sulfidebased metabolism may have preceded the present, oxygen-based life on the planet by billions of years (7)(8)(9).
In sharp contrast to its pivotal role early in the evolutionary timeline, H 2 S is known mostly for being a foul smelling poisonous gas, associated with sewers, septic tanks, and as a weapon of chemical warfare during the First World War. Consequently, majority of the research pertaining to this gas has been conducted from a toxicology point of view (10,11). With studies published as long back as 1803 highlighting the detrimental effect of H 2 S on animals, along with the recent gene expression data, H 2 S was condemned as a respiratory and metabolic poison (12). It was not until the 1940s that the "transsulfuration" pathway involving the production of H 2 S by interconversion between cysteine, homocysteine via cystathionine was described for the first time in liver homogenates (13,14). Further studies led to detailed biochemical characterization of the enzymes cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE) involved in the transsulfuration reaction. Later, another enzyme, 3-mercaptopyruvate sulfurtansferase (3-MST) was identified as a part of H 2 S biogenesis pathways (15)(16)(17)(18). However, the functional implication of the H 2 S biogenesis remained elusive for a long time. First glimpse of H 2 S involvement in cellular physiology emerged from the studies demonstrating measurable levels of endogenous H 2 S within brain tissues of healthy individuals (0.65-0.73 mg/g) and animals (1.57 ± 0.04 μg/g) (19)(20)(21). Along these lines, higher levels of neuronal H 2 S was found to be due to greater expression of CBS in the brain tissues. Additionally, H 2 S production in the brain tissue was efficiently reduced using pharmacological CBS inhibitors (hydroxylamine and aminooxyacetic acid). Further studies proposed that H 2 S facilitates the induction of hippocampal long-term potentiation (LTP) by enhancing the activity of N-methyl D-aspartate (NMDA) receptors (22). Later, H 2 S was found to relax vascular smooth muscle by activating ATPsensitive K + , intermediate conductance Ca 2+ sensitive K + , and small conductance Ca 2+sensitive K + channels (23)(24)(25). Importantly, H 2 S was identified to protect from oxidative stress and ischemia-reperfusion injury by multiple mechanisms such as restoring the levels of GSH and direct scavenging of mitochondrial ROS ( Fig. 1) (26,27). These discoveries further led to the disclosure of mechanisms by which H 2 S protects various organs, including the heart and kidney from oxidative stress and ischemia-reperfusion injury (28). Based on these studies, H 2 S was inducted as the newest member of the family of small molecule gaseous transmitters or "gasotransmitters" alongside nitric oxide (NO) and carbon monoxide (CO) (29). Along this line hydrogen gas (H 2 ) has also emerged as a potential gaseous signaling molecule with therapeutic antioxidant function (30,31). Recently, H 2 S was found to have a protective role in airway epithelial cells infected with respiratory syncytial virus (RSV) demonstrating for the first time that this molecule might be used as a therapeutic agent. Needless to say, the role of H 2 S has permeated to areas of metabolism, redox physiology, neurophysiology, apoptosis, angiogenesis, ageing, inflammation, atherosclerosis, pulmonary diseases among others with a whole spectrum of physiological implications (32)(33)(34).
H 2 S producing bacteria were discovered way back in 1877. Many investigators demonstrated bacterial production of H 2 S by its rotten egg smell and its ability to react with lead acetate resulting in the blackening of paper strips impregnated with lead acetate (12,35). In fact, lead acetate test was successfully exploited to distinguish between the paratyphoid and enteritidis groups and still remains an indispensable diagnostic tool (36). In the area of marine microbiology, H 2 S emitted from deep sea vents is often referred to as the "sunlight of the deep ocean" (37,38). H 2 S forms an important source of metabolic energy for the microorganisms inhabiting such niches, reminiscent of the primordial earth (6). Many bacterial species were demonstrated to possess orthologs of the transsulfuration pathway enzymes (CBS and CSE) and of 3-MST (36,(39)(40)(41). However, the significance of H 2 S biogenesis in bacteria remained poorly characterized. In 1960s, co-culture experiments with Desulfovibrio desulfuricans/Pseudomonas aeruginosa and Escherichia coli/Staphylococcus aureus provided the first evidence of a possible "protective" role of H 2 S in bacteria. H 2 S produced by D. desulfuricans was demonstrated to be the "diffusible" factor responsible for imparting pseudomonads the ability to resist heavy metal (e.g., mercury) toxicity (42). Similarly, H 2 S produced by E. coli protected S. aureus from merbromin and mercuric chloride (43). Surprisingly, it took more than four decades to discover additional roles of H 2 S in protecting diverse bacterial species from oxidative stress and antibiotics (44). Similar reports of protective influence of H 2 S have emerged in plants and nematodes, however, they are beyond the scope of this review. Altogether, it appears that H 2 S is an important biological effector molecule with diverse roles in organisms ranging from bacteria to mammals.
While there has been a steady rise in the number of studies pertaining to the physiological role of H 2 S in mammalian systems, there is a significant lack in our understanding of the same when it comes to infectious agents like bacteria and viruses. A survey of PubMed also shows that the studies on H 2 S and bacterial/viral infections are relatively fewer as compared to mammalian systems (Fig. 2). Keeping this in mind, our effort is to compile a summary of the existing studies providing insights on how H 2 S exerts its biological influence on infectious diseases caused by bacterial and viral agents. First, we will provide a brief description of H 2 S biogenesis pathways and chemical properties of H 2 S. This will be followed by a description of studies on the contribution of H 2 S in influencing the physiology of bacteria and virus infected cells.
Apart from transsulfuration, an additional pathway also exists which leads to the biogenesis of H 2 S. Cysteine aminotransferase (CAT, EC 2.6.1.3) catalyses the reaction of cysteine with keto acids (e.g., α-ketoglutarate) to form 3-mercaptopyruvate, which is subsequently desulfurated by 3-mercaptopyruvate sulfurtransferase (3-MST, EC 2.8.1.2) to form H 2 S (Fig. 3) (45). More recently, a new pathway to generate H 2 S using D-cysteine has been identified. The enzymes D-amino acid oxidase (DAO) along with 3-MST carry out biogenesis of H 2 S from D-cysteine (49). Nonenzymatic reactions also lead to the generation of H 2 S inside cells, however, their contribution is minor and remain poorly understood and characterized.

Cystathionine Beta Synthase
Human cystathionine beta synthase (CBS) is a tetramer and is allosterically stimulated by Sadenosyl methionine (SAM) which binds to a conserved "CBS pair domain" in the Cterminal end of the protein (50,51). CBS catalyses the first and committed step of the transsulfuration pathway which canonically, leads to the production of cystathionine from serine and homocysteine (52). However, when serine is replaced by cysteine, H 2 S is produced (Fig. 3). CBS can generate H 2 S by additional reactions including β replacement of cysteine by water to form serine and β replacement of cysteine by a second molecule of cysteine to form lanthionine. In kinetic terms, the β replacement of cysteine with homocysteine remains the most favorable (52). As mentioned earlier, CBS utilizes PLP as a cofactor and is a type-II PLP binding protein (53). The cofactor remains covalently linked to the active site lysine via Schiff base formation and is pertinent to the enzymatic activity of the protein. In addition to PLP, human CBS also contains heme which acts as a redox dependent gas sensor (54). Apart from this the heme moiety also functions as a "metabolic switch" committing the pathway toward H 2 S production (55). Under ER stress, heme oxygenase is induced, which catabolises heme in presence of molecular oxygen to produce biliverdin and CO, the later one binds to heme cofactor of CBS and inhibits its activity. This inhibition leads to low levels of cystathionine and increased levels of homocysteine. These metabolites cue the second enzyme, CSE, to increase the production of H 2 S from cysteine and homocysteine further highlighting the metabolic flexibility of this pathway (55). cysteine in the active site. This leads to the formation of a bound persulfide which acts as a source of H 2 S under reducing conditions or in presence of acceptors like thioredoxin. 3-MST is localized to the mitochondria unlike CBS and CSE which are cytosolic, where it is believed to contribute bioenergetically via sulfide oxidation. Also, unlike the transsulfuration enzymes, 3-MST is inhibited under oxidizing conditions due to a labile active site cysteine which gets converted to cysteine sulfonate leading to enzyme inactivation (57).
MST/DAO Pathway-In addition to L-cysteine, H 2 S production was observed in brain homogenates when D-cysteine was used as a substrate. This led to the discovery of new pathway involving peroxisomal enzyme D-amino oxidase (DAO) in H 2 S biogenesis. Dcysteine is metabolized by DAO to 3-mercaptopyruvate (3MP), which then translocates to mitochondria where it is converted to H 2 S and pyruvate. It has been reported that the production of H 2 S from D-cysteine is ~ 60 times greater in comparison to L-cysteine. Since DAO is only localized to the brain and the kidney, the functionality of the 3MST/DAO pathway for the production of H 2 S is only relevant to these tissues (49,58).

Chemical Biology of H 2 S
H 2 S gas was discovered in 1777, by Carl Wilhelm Scheele, and was largely considered as a toxic gas for over hundred years (59). Based on toxicological studies, the permissible exposure limit of H 2 S is 10 ppm and 800 ppm exposure for 5 min is the lethal concentration for 50% of humans (LC50) (60,61). Much of its toxicity is owed to the fact that H 2 S is known to inhibit respiration thereby acting as a metabolic poison. When present at higher concentrations it causes reversible inhibition of cytochrome c oxidase (complex IV), thereby perturbing mitochondrial respiration and oxidative phosphorylation (46). This is further exemplified by the observations that H 2 S induces a state of "suspended animation" with consequent lowering of metabolic rate and body temperature in mice (61).
The standard two electron redox potential of H 2 S/S 0 couple, −0.23 V (noted +0.140 value in acidic condition) at pH 7 (versus the standard hydrogen electrode) is comparable to that of major cellular antioxidant buffers, glutathione disulfide/glutathione (E°′= −0.262 V) and cystine/cysteine (E°′= −0.245 V) redox potentials (62)(63)(64)(65). While the physiological concentration of H 2 S is a matter of ongoing controversy, it seems that low nM concentrations are most likely (66). Only in case of aorta, the reported concentration of free H 2 S is ~ 20-100 fold higher than that of other tissues (67). Interestingly, the flux of sulfur into H 2 S is comparable to that of GSH, indicating that the low nM levels are maintained as a consequence of higher sulfide clearance rate (59,68). The low steady-state concentration of H 2 S than GSH (~10 mM) precludes its involvement in counteracting oxidative stress by acting as an antioxidant buffer (46). Alternatively, it is proposed that H 2 S can modulate intracellular redox signaling by modifying cysteine thiols of the various cellular proteins (Spersulfidation) coordinating redox homeostasis. However, since a direct reaction of H 2 S with thiols is unlikely, the mechanism by which persulfides are formed intracellularly is poorly understood (69,70). H 2 S is lipophilic and is known to permeate freely through biological membranes without any assistance from membrane channels (lipid bilayer permeability P M ≥ 0.5 ± 0.4 cm/s) (71). Being a weak acid, it dissociates immediately and equilibrates with its anion HSand S 2in aqueous solution as shown in Eq. (1).
The pK a1 for H 2 S dissociation ranges from 6.97 to 7.06 at 25 °C, while pK a2 is estimated to be between 12.20 to 15.00 at 25 °C (72). Based on these values it is calculated that the ratio of HS -:H 2 S is 3:1 at physiological pH of 7.4 (72). Nevertheless, total intracellular H 2 S levels is referred to as total free sulfide pool (i.e., H 2 S + HS -+ S 2-). Based on its chemical features, H 2 S can influence cellular redox physiology via four mechanisms: (1) scavenging of ROS and RNS, (2) reaction with metal centres, (3) modulation of cellular respiration, and (4) reaction with protein cysteine thiols to generate persulfides (S-persulfidation-an oxidative posttranslational modification [oxPTM]) (69,73). These mechanisms are described in the following section.

H 2 S as a Free Radical Scavenger
H 2 S acts as a cytoprotective molecule and has the ability to directly scavenge free radical species (74). Owing to its nucleophilic properties, H 2 S has been shown to react with oxygen (O 2 ), ROS, peroxynitrite (ONOOH/ONOO -), and hypochlorite (HOCL/ -OCL) (65,75). The apparent second-order rate constants of H 2 S with various oxidants have been summarized in Table 1. While these studies indicate a direct scavenging of oxidants by H 2 S in vitro, the low concentrations of H 2 S (10 nM to 3 μM) compared to other antioxidants (GSH; 1-10 mM) in vivo raised substantial concerns about its role in remediating ROS/RNS under biologically relevant conditions (66,(76)(77)(78)(79). Alternatively, H 2 S has been shown to increase GSH production by enhancing the inward transport of cystine and inducing the expression of GSH-biosynthetic enzyme, GCL (γ-GCS) (80,81). This increase in intracellular GSH could be another mechanism by which H 2 S indirectly participates in protection from oxidative stress.

Reaction with Metal Centers
The interaction of H 2 S with metals falls into two categories: (i) electron-transfer reaction and (ii) coordinate complex formation (65). In the first category, complete electron transfer occurs between the sulfide species and the metal, whereas coordinate complex formation involves binding of the sulfur species to the metal ligand (65). These reactions are predicted on the basis of chemical properties of H 2 S to act as a nucleophile. Interestingly, a wine-like model was used to study the reaction mechanism of metals with H 2 S. Sulfidic off-odors encountered during wine production are due to the presence of H 2 S and low-molecularweight thiols (82). These off-odors are usually removed in a process called Cu fining, wherein Cu (II) is added to selectively and rapidly form ~1.4:1 H 2 S/Cu and ~2:1 thiol/Cu complexes, resulting in oxidation of H 2 S and reduction of Cu (II) to Cu (I) (82). The CuS precipitate formed is than subsequently removed from the wine by racking and/or filtration (82).  (86). The K i for this reaction is 0.2 μM with purified CcO (86). Most of the studies demonstrating inhibitory effect of H 2 S on respiration via interaction with CcO were done using very high/nonphysiological concentrations of H 2 S. However, it was shown that the liver mitochondria of H 2 S treated rats show biphasic respiration profile (87). Low concentrations (0.1-3.0 μM) of H 2 S induces respiration whereas higher concentrations (30-100 μM) inhibits (87). At lower concentrations, H 2 S acts as a mitochondrial electron donor and stimulates electron transport chain (87,88).
Other than CcO, H 2 S is known to covalently modify ferryl/peroxo heme within hemoglobin and myoglobin resulting in the formation of green colored sulfhemoglobin and sulfmyoglobin species, both of which are indicators of H 2 S poisoning (89). Additionally, H 2 S can react with nonheme iron present in iron-sulfur cluster containing proteins to generate insoluble precipitates (65). Lastly, H 2 S is reported to react with a copper-containing protein (Cu-Zn-SOD) (90). This reaction involves copper-catalysed reduction of O 2 − to H 2 O 2 Oxidation of H 2 C to S 0 (90).

H 2 S and Cellular Bioenergetics
The effects of H 2 S on cellular bioenergetics are largely derived from examining mitochondrial function. The effect of H 2 S on mitochondria is complex, exhibiting two opposing effects; inhibition and stimulation of mitochondrial bioenergetics (88). Oxidation of H 2 S by mitochondrial inner membrane localized Sulfide-Quinone oxidoreductase (SQR) leads to transfer of electron from H 2 S to ubiquinone and increases the flux of electron transport to mitochondrial respiratory complex III and IV, thereby leading to enhanced oxygen consumption and cellular respiration (91,92).
Recently, it was demonstrated that H 2 S has a biphasic effect on cellular oxygen consumption/mitochondrial electron transport (87,88). These investigators creatively adapted Seahorse XF technology to precisely measure dynamic changes in mitochondrial bioenergetics in real-time in response to a gradient of H 2 S. Interestingly, treatment of isolated mitochondria with low H 2 S concentrations, NaHS (<1 μM) enhances mitochondrial oxygen consumption rate (OCR), ATP turnover rate and leads to increased maximal respiratory capacity (87). In contrast, treatment with high concentrations of NaHS (30-300 μM) causes reduced mitochondrial OCR and ATP generation, which is consistent with previous studies showing the inhibitory effect of H 2 S on mitochondrial respiration when present at high concentrations (84,93). The low endogenous concentrations of H 2 S in mitochondria is primarily maintained by mitochondrial localized 3-Mercaptopyruvate sulfurtransferase (3-MST) (87). Supplementation of isolated mitochondria with 3mercaptopyruvate (3-MP) leads to enhanced H 2 S production by 3-MST pathway and induces mitochondrial bioenergetic parameters (87). Subsequently, genetic silencing of 3-MST leads to reduced basal OCR and due to absence of 3-MST there is no stimulatory effect of 3-MP on mitochondrial bioenergetic parameters (87).
Furthermore, it was found that the stimulatory effect of 3-MP on mitochondrial bioenergetic parameters was absent in mitochondria isolated from aged mice as compared to young mice (88). Moreover, exposure of H 2 S was shown to cause physiological alterations which enhanced thermotolerance and life span of Caenorhabditis elegans (94). The other two PLPdependent cytosolic enzymes CBS and CSE also maintain endogenous H 2 S levels. Under stress condition, CSE is known to translocate into mitochondria and stimulate mitochondrial H 2 S generation. This subsequently results in an increased ATP generation and resistance to hypoxia (95). Furthermore, elevated endogenous H 2 S in colon cancer cells have been shown to regulate cell migration and invasion (96). Additionally, pharmacological inhibition of H 2 S production diminished the growth of cancer cells by suppressing basal respiration, ATP production, spare respiratory capacity, and glycolysis (96,97).

Protein Persulfidation as a Mechanism of H 2 S Mediated Signaling
H 2 S exerts its signaling property via oxidative posttranslational modification (oxPTM) of cysteine residues, called S-persulfidation (98)(99)(100). Persulfidation (R-SSH) is the oxidation of thiol (-SH) from −2 to −1 oxidation state (101). Being a reductant, H 2 S/HScan carry out the persulfidation reaction only when one of the reagents (-SH group of proteins or H 2 S) is in the oxidized form (101). The proposed reactions for the formation of S-persulfide bond by the nucleophilic attack of HSanion on reversibly oxidized protein -SH group is shown in (Fig. 4).
Recently, it was reported that persulfidation protects proteins from oxidative stress-induced damage and the over oxidized persulfidated cysteine sulfonic acid (P -SSO 3 2 − ) can be reversed to thiol (-SH) by the depersulfidase activity of thioredoxin (102). Thus, persulfidation can act as a protective mechanism against oxidative stress-induced protein damage (102). Using LC-MS-based techniques, S-persulfidation of proteins involved in fatty acids and carbohydrate metabolism, cellular response to stress, cell redox homeostasis, translation, and cell cycle were identified (73). Similarly, H 2 S was shown to modify cysteines in about 10-25% of liver proteins including actin, tubulin, and glyceraldehyde-3phosphate (GAPDH) by persulfidation under physiological conditions and modulate their activities (103). Several key host transcription factors, Nrf2 and Nf-κB, are directly targeted by H 2 S via S-persulfidation (98). Apart from eukaryotic systems, a recent study, for the first time, assessed widespread S-persulfidation of proteins in Staphylococcus aureus (S. aureus) (104). More importantly, H 2 S exposure increased the level of S-persulfidation, whereas mutations in transsulfuration pathways (cysM and metB) had an opposite effect (104).
Lastly, this study revealed extensive persulfidation of transcription factors involved in virulence regulation (SarA family) and interaction with host immune response (superantigen-like proteins [SSLs]) (104).

Detection of H 2 S and Protein S-Persulfidation Tools for Detection and Quantitation of H 2 S
To achieve a better understanding of the physiological effects of H 2 S, it is imperative to determine the levels of this gasotransmitter in its free and other biological forms. This becomes especially important as the role of H 2 S as a protectant versus poison has been Europe PMC Funders Author Manuscripts contended throughout the course of its study as a biological effector molecule. The suitability of any detection method relies heavily on three pertinent aspects namely sensitivity, reproducibility and experimental ease. A multitude of detection methodologies are available for qualitative and quantitative measurement of H 2 S levels. While this may seem advantageous at first glance, it has led to a huge variability in the reported levels of "bioavailable" H 2 S throughout literature. Furthermore, additional factors influencing detection arise due to experimental aspects like biological sample (tissue, sera, and cells), pH, and oxygen. The latter have been demonstrated to exert a direct effect on the stability of H 2 S (105).
The detection methods range from simple colorimetric assays to techniques like gas chromatography (GC), High pressure liquid chromatography (HPLC), electrochemical, polarographic, fluorescent, and recently developed nanotechnology-based systems. Some of these techniques are summarized in Table 2. Although these methods have been widely used for biological H 2 S detection, they are not devoid of pitfalls. The issues of sensitivity, invasiveness, artefactual readout, half-life, stability, permeability, lack of spatio-temporal insight, and cumbersome experimental setup cannot be overlooked. Therefore, the reporting and interpretation of experimental data becomes immensely influenced by the method that a researcher chooses to adopt. Many authors have argued that for a dynamic gaseous effector molecule like H 2 S, the absolute numbers in terms of concentration may actually not matter as much as the determination of the qualitative trend of its rise and fall under different physiological conditions. This may, however, not hold true for pathophysiological conditions wherein accurate determination of H 2 S levels may become indispensable for diagnosis of certain diseases.
Apart from the aforementioned techniques, recent studies using H 2 S sensitive fluorescent proteins as genetically encoded biosensors have garnered immense interest. GFP molecule has been reengineered to contain the unnatural amino acid p-azidophenylalanine (pAzF).
This azido group can be reduced specifically by H 2 S imparting selectivity to the GFP molecule, now termed as hsGFP. An orthogonal tRNA-tRNA synthetase system from E. coli was used for the selective incorporation of pAzF into hsGFP molecule in response to the Amber (TAG) codon. In presence of both H 2 S and pAzF, the chromophore p-azidobenzylideneimidazolidone of hsGFP is converted to p-aminobenzy-lideneimidazolidone with fluorescence excitation and emission maxima at 454 nm and 500 nm, respectively. This genetically encoded probe has been used in mammalian cell line HEK293T for the detection of intracellular H 2 S. Such a genetically encoded biosensor is noninvasive and can reflect the dynamic nature of the turnover of H 2 S inside cells (128). Furthermore, such sensors can be targeted to specific cellular locations. Taken together, a genetically encoded biosensor appears to be a promising tool to study the levels of H 2 S under physiological conditions.

Tools for Detection of Protein S-Persulfidation
In addition to H 2 S, the detection of the post translational modification caused by it is also of the utmost importance to fully appreciate the functional relevance of this gasotransmitter. Protein persulfidation was identified to be the mechanism by which H 2 S exerts its signaling function (103). Detection of this modification, however, poses a significant challenge as the persulfide group exhibits similar reactivity to free thiols (129). Table 3 summarizes some of the widely used methods for the detection of intracellular protein persulfidation levels.

Role of H 2 S in Bacterial Physiology
While the initial co-culture experiments described earlier provided a valuable clue with regard to potential of H 2 S in protecting bacteria from toxic compounds, in depth examination of these findings was never attempted (42,43). It was only in 2011 that a study highlighted the importance of H 2 S in protecting bacteria from antibiotics and oxidative stress (44). In this context, H 2 S has been termed as a "double edged sword" mitigating not only the effects of antibiotics but also the resulting oxidative stress caused by them. To ascertain the role of H 2 S in E coli, the authors compared wild type and 3-MST deficient E. coli by a phenotypic microarray. While these strains showed no difference with respect to growth defects in vitro, the 3-MST deficient strain became highly susceptible to structurally and functionally different classes of antibiotics. Similar results were obtained for CBS/CSE deficient strains of P. aeruginosa, S. aureus, and B. anthracis, establishing the protective role of H 2 S across gram negative and gram-positive bacteria. Overexpression of 3-MST led to enhanced protection against spectinomycin whereas chemical inhibition on 3-MST, CBS, and CSE rendered them highly susceptible to a variety of antibiotics. NaHS, an H 2 S donor chemically complemented these mutant strains establishing the role of endogenously generated H 2 S as a protective mechanism against antibiotics.
Several studies have shown that a wide range of antibiotics exert killing by triggering ROS generation in addition to inhibiting the function of their primary targets (135,136). Antibiotics have been shown to stimulate respiration, which increases generation of toxic hydroxyl radicals via Fe 2+ -catalysed Fenton reaction (137). Consistent with these observations the authors have shown that pretreatment with Fe-chelator (dipyridyl) or ROS scavenger (thiourea) induces gentamycin resistance to both the wild type and H 2 S deficient strains of E. coli. Interestingly, a similar degree of protection from gentamycin was observed when the cells were treated with NaHS. Additionally, all the H 2 S deficient mutant strains exhibited severe susceptibility to H 2 O 2 which was mitigated when they were pretreated with NaHS. DNA damage is one of the direct consequences of oxidative stress generated by antibiotics (138,139). On treatment with sublethal levels of ampicillin, which is known to cause oxidative stress, 3-MST deficient strain of E. coli showed tell-tale signs of DNA damage. Overexpression of 3-MST and pretreatment with NaHS ameliorated this damage. Additionally, the antioxidant effect of H 2 S was also shown to be in part due to the stimulation of antioxidant enzymes such as catalase and superoxide dismutase (SOD  (Fig. 5).
Apart from the above studies, another mechanism was put forward to explain the protective role of H 2 S. This involved the master regulator CysB which is involved in regulation of a number of sulfur metabolism genes in E. coli. CysB regulates the expression of TcyP, a cystine importer. During oxidative stress, H 2 O 2 interacts with cysteine leading to its depletion and induction of CysB regulon including TcyP. As a consequence, TcyP leads to an increased influx of cystine/cysteine inside the cytoplasm resulting in the enhanced production of H 2 S via 3-MST. More recently, using chemical-biology approaches, we developed a series of bacteria specific H 2 S donors to explain the mechanism of H 2 S mediated protection (141). On pretreatment of E. coli with one such H 2 S donor (1c), we showed increased resistance to bactericidal antibiotics (ampicillin and amikacin) and H 2 O 2 . Furthermore, using a noninvasive biosensor of cytoplasmic redox potential (roGFP2) (142,143), we for the first time, precisely measured real-time changes in the redox physiology of E.coli in response to antibiotics in the presence or absence of H 2 S.
Importantly, we showed that while elevation in endogenous H 2 S levels does not influence redox physiology of E.coli, it efficiently reversed antibiotics induced oxidative shift in the cytoplasmic redox potential of bacteria. To further probe mechanistic aspects of these findings, we discovered a functional association between H 2 S-directed cytoprotection and alternate mode of cellular respiration catalysed by cytochrome bd oxidase (CydB). H 2 S, due to its strong affinity for metals such as copper, is known to inhibit copper-heme containing cytochrome bo oxidase (CyoA). Under these conditions, the respiration proceeds via a less energy efficient CydB. In agreement with this, treatment of the cells with 1c led to a downregulation of cyoA transcript, whereas the transcripts of alternate respiratory oxidases such as cydB and appY were either maintained or enhanced, respectively. This realignment of respiratory oxidases mimics the expression profile of E. coli grown under respiratory arrest conditions (e.g., hypoxia), implicating H 2 S in respiration inhibition and metabolic slow down. In contrast, ampicillin treatment enhanced the expression of the energy efficient cyoA and repressed cydB, consistent with the reported hyperactivation of electron transport chain by bactericidal antibiotics (144). However, pretreatment of the cells with 1c reversed the influence of ampicillin on cyoA and cydB transcripts. In agreement with these findings, the cyoA mutant pretreated with 1c remained protected from ampicillin toxicity, whereas 1cderived H 2 S remained completely ineffective in protecting cydB mutant. The cydB from E. coli has also been shown to reduce H 2 O 2 by acting as catalase and quinol peroxidase (145

Oxidative Stress is an Integral Part of Infection with Diverse Class of Viruses
Oxidative stress has been linked to vast group of etiological agents that cause acute and chronic diseases such as infection with viruses, bacteria, and parasites (149). Viral and bacterial infections in particular have been linked to induce ROS/RNS production, alteration in metabolic pathways, and leading to several disease associated complications (149,150). However, this field still lacks mechanistic insight. The impact of these infectious agents on host redox physiology and how this could be targeted for therapeutic benefits remains a challenging area of research.
In case of viral infections, induction of oxidative stress inside host is a prerequisite for successful infection and long term viral replication. RNA viruses such as influenza and paramyxovirus infection generates ROI via activation of monocytes and polymorphonuclear leukocytes (149). A study indicates that the oxidative state of the host cells provides an environment permissive for viral replication (151 The role of oxidative stress has been extensively studied in retroviruses such as HIV-1 (Human Immunodeficiency Virus). Studies have shown that HIV-1 replication induces ROS generation and decreases cellular antioxidants such as GSH and Trx, and modulates immunopathogenesis during AIDS progression (155,156). Plasma and peripheral blood mononuclear cells (PBMCs) of AIDS patients show reduction in concentration of other major antioxidants like cysteine, methionine, vitamins C and E, along with elevated levels of lipid peroxidation products (157). At molecular level, ROS has been shown to induce the activity of redox sensitive transcription factors Nf-κB, AP-1, and Sp1, which regulates HIV-1 gene transcription by binding to 5′-LTR promoter (158,159). Despite these studies, for a very long time, the relation between HIV-1 and oxidative stress remained circumstantial. This was largely owing to the lack of sophisticated and sensitive tools to measure intracellular redox potential of HIV-1 infected cells during various stages of infection. To fill this knowledge gap, we exploited a noninvasive biosensor of GSH redox potential (Grx1-roGFP2; E GSH ) and accurately measured oxidative stress in the cytoplasm and mitochondria of HIV-1 infected monocytes (160). We demonstrated that monocytes latently infected with HIV-1 are intrinsically resistant to oxidative stress and displayed reductive E GSH (160). More importantly, we showed that a marginal oxidative shift in E GSH (25 mV) triggers reactivation of HIV-1 without adversely affecting cellular physiology. Furthermore, supplementation with antioxidants such as N-acetylcysteine kept HIV-1 in a silent state by preventing an oxidative shift in E GSH required for viral activation (160).
Lastly, global expression analysis revealed that pathways associated with redox metabolism are significantly affected during HIV-1 latency and reactivation (Fig. 6) (160). Taken together, these results highlight the central role of host redox physiology in modulating HIV-1 life cycle.
At molecular level HIV-1 infection or exposure to HIV-1 related proteins downregulates the Nuclear factor-erythroid-2 p45 related factor 2-Antioxidant Response Element (Nrf2/ARE) pathway, leading to reduced expression of antioxidant genes (161,162). Nrf2 is a constitutive transcription factor and master regulator of the antioxidant response (163,164). Nrf2 is inhibited in cytosol by Kelch-like ECH-associated protein-1 (Keap1), which is a redoxsensitive ubiquitin ligase substrate adaptor leading to ubiquitination and degradation of Nrf2 (165). Interestingly, Nrf2 inducer (Sulforaphane) has the ability to block HIV-1 infection in primary macrophages, which are the long-lived reservoirs of HIV-1 in infected individuals (166). In this direction, sulforaphane has also been shown to enhance phagocytic activity of HIV-1 infected monocytes-derived macrophages (MDMs) and alveolar macrophages (Ams) from HIV-1 transgenic rats, thereby reducing the severity of HIV-1 related pulmonary dysfunctions (161). Nrf2 activation has also been shown to assist Marburg virus ((-ss) RNA) (MARV), Kaposi's sarcoma-associated herpesvirus (dsDNA) (KSHV), and Dengue virus (( + ss) RNA) replication. Activation of Nrf2 induced by VP24 and vFLIP proteins of MARV and KSHV, respectively, leads to dysregulation of host antiviral response and modulates viral gene expression, thereby ensuring a conducive environment for infection and also promotes the survival of infected cells (167,168). Dengue infection induced oxidative stress has been shown to activate Nrf2 thereby modulating the level of oxidative stress and affecting both antiviral and cell death response (169). While these studies clearly establish a connection between oxidative stress and infection with pathogenic viruses, the contribution Europe PMC Funders Author Manuscripts of H 2 S in these process is poorly understood. Interestingly, Nrf2/Keap-1 pathway could provide the missing link between H 2 S, redox stress and virus infections. H 2 S has been shown to inhibit Keap1 by persulfidation of Cys 151, which leads to translocation of Nrf2 into the nucleus and its subsequent binding to Antioxidant Response Element (ARE). This results in the induction of genes encoding antioxidant and phase II detoxifying enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase-1 (NQO-1), glutamate cysteine ligase catalytic subunit (GCLC), and thioredoxin reductase-1 (TXNRD-1) (163,165). In relation to this H 2 S has been shown to protect against cellular senescence and oxidative stress via S-sulfhydration of Keap1 and resulting activation of Nrf2 (165). Further experimentation on H 2 S biogenesis and Nrf2/ARE pathway during viral infections will provide next stage of insight in this process. The following section provides recent knowledge in this direction.

H 2 S and Host-Pathogen Interaction
Host-derived H 2 S has a marked effect on the outcome of bacterial and viral infections.
Blocking the host transsulfuration pathway in macrophages by propargylglycine increased the viability of Mycobacterium smegmatis. This impairment in bacterial clearance was shown to be due to defects in the phagolysosomal fusion during infection. Treatment with Nacetylcysteine (NAC), which is known to increase cysteine flux through H 2 S biogenesis pathway, significantly increased the phagolysosomal fusion resulting in vacuolar acidification and killing of mycobacteria (170). H 2 S was found to inhibit the induction of an inflammatory response on infection with Mycoplasma fermentans. Underlying mechanism revealed that H 2 S inhibited the activation and nuclear translocation of a redox sensitive transcription factor NF-κB, thereby diminishing the transcription of proinflammatory genes (171,172). One of the mechanism by which H 2 S affects the activity of a global transcriptional regulator, NF-κB, is by persulfidation of Cys-38 residue in the p65 subunit (131). H 2 S production by gut microbiota presents itself as an interesting line of study to explore the effect of bacteria-derived effector molecules affecting host physiology and pathophysiology. Sulfide reducing bacteria (SRB) represent a major class of the normal gut microbiota (173). The predominant genera residing in gut are Desulfovibrio, Desulfobacter, Desulfolobus, and Desulfotomaculum. SRBs are the major contributors of nonenzymatic H 2 S produced in the human body (174). Some of the initial studies to explore the significance of gut microbiotaderived H 2 S came from experiments done on germ free animals. It was observed that fecal samples of germ free mice contained half the H 2 S as compared to control mice (175). In addition to this, free H 2 S levels in inferior vena cava, blood plasma and in gastrointestinal tissues, were shown to be diminished in germ free mice. Apart from this, sulfane sulfur levels of plasma, adipose, and lung tissues were also found to be lower in such mice. This implicated the gut microflora as a potential source of circulating H 2 S for the host (176).
Recent studies have also shown that colonocytes are capable of using H 2 S as an energy source (177).
Gut bacteria-derived H 2 S has been shown to have both protective and detrimental effects on colonic health. Increased fecal sulfide levels have been found in patients with ulcerative  (178). Furthermore, it has been suggested that epithelial damage associated with UC is due to increased availability of dietary sulfate for SRBs (179). In contrast, using animal models of colitis, it was demonstrated that scavenging bacterial H 2 S by bismuth did not ameliorate the symptoms of colitis (180). In fact, the condition was shown to improve on exogenous H 2 S administration (181). The ability of luminal H 2 S to modify secreted defensive proteins like trefoil factor 3 (TFF3) by reduction of the disulfide bond is believed to be a potential mechanism of the anti-inflammatory role of H 2 S. TFF3 plays an important role in mucosal repair and regeneration (182).
Our survey of literature revealed the existence of very few reports on H 2 S and viral infections. Existing studies have highlighted the role of H 2 S in modulating Respiratory Syncytial Virus (RSV) (( -ss) RNA virus) infection (183). RSV causes lower and upper respiratory tract infection in infants for which there is no vaccine and only limited supportive measures for treatment exist with no real benefits (184). RSV mediates its influence on the host by upregulating the expression of various Nf-κB and IRF-3 dependent cytokines and chemokines (185). This leads to inflammation, cellular infiltration in lungs and other pulmonary dysfunctions. Importantly, RSV infection resulted in downregulation of expression and impaired activity of H 2 S biosynthesis enzymes. As a consequence, the endogenous levels of H 2 S were diminished in RSV infected cells (183). Pharmacological inhibition or genetic silencing of CSE (cystathionine gamma-lyase) enhances RSV multiplication and exacerbates disease condition, airway dysfunction, and pulmonary inflammation (186,187). Consistent with these findings, exogenous administration of H 2 S reduces the secretion of viral induced chemokines and cytokine through inhibition of NF-κB mediated activation of genes encoding proinflammatory cytokines (183). H 2 S treatment (using slow releasing H 2 S donor-GYY4137) significantly blocked RSV replication in vitro and in vivo by targeting viral assembly, release, viral spread, and replication ( Fig. 6) (183,186). Moreover, H 2 S treatment significantly improved clinical disease parameters and pulmonary dysfunction on RSV infection (186). Similarly, H 2 S also exerts antiviral and antiinflammatory effects on the viruses in the family of Paramyxoviridae; human metapneumovirus (hMPV) and Nipah virus (NiV) (183).
H 2 S has also been shown to affect replication of highly pathogenic enveloped RNA virus from Ortho-, Filo-, Flavi-, and Bunyavirus families ( Fig. 6) (189). H 2 S was shown to significantly reduce replication of all the above families of viruses (188). As explained earlier, studies confirmed that H 2 S targets the transcription factor Nf-κB and IRF-3 nuclear translocation to inhibit the release of viral induced proinflammatory mediators (188).
Lastly, H 2 S has been recently shown to modulate Coxsackie virus B3 (CVB3) infection induced inflammatory response, which is a predominant cause of human myocarditis and ultimately leads to heart failure (190). Treatment of CVB3 infected rats with H 2 S significantly resulted in downregulation of proinflammatory mediators, reduces myocardial injury, and alleviates damage of myocardial cells (191). H 2 S was shown to inhibit Nf-κB Europe PMC Funders Author Manuscripts signaling by lowering IκBα degradation leading to reduced nuclear translocation and DNA binding ability of Nf-κB (191). CVB3 infection also induces MAPK signaling cascade by activating ERK1/2, p38 and JNK1/2 which are upstream signaling molecule involved in activation of Nf-κB (192). H 2 S treatment also showed reduced CVB3 induced activation of ERK1/2, p38, and JNK1/2 in rat myocardial cells, thereby supressing the expression of inflammatory mediators and alleviating myocardial damage (191).   Pathways involved in enzymatic biogenesis of H 2 S. The transsulfuration pathway consisting of the enzymes cystathionine β-synthase (CBS) and Cystathionine γ-lyase (CSE) is the major pathway for biological H 2 S production. In addition to this 3-mercaptopyruvate sulfurtransferase/cysteine aminotransferase (3-MST/CAT) pathway also contribute significantly to the production of H 2 S.       Table 2 Tools for detection and quantification of H 2 S

Detection method Description References
Colorimetric H 2 S reacts with metal salts like lead acetate, bismuth chloride, silver nitrate to form the lead sulfide which can be detected and quantified using UV-VIS spectroscopy. (106)(107)(108) "Zinc trap" method in which zinc acetate reacts with H 2 S forming zinc sulfide with subsequent acidification with N, N-dimethyl phenylenediamine. The product can be detected and quantified using UV-VIS spectroscopy. (109,110) Chromatographic Gas chromatography has been combined with flame photometric detectors, ion chromatography, silver particle trapping, and chemiluminescent detectors. (20,67,(111)(112)(113)(114) HPLC of sulfide derivatized with monobromobimane, dibromobimane, pphenylenediamine, and Fe 3+ . Reverse Phase HPLC of methylene blue formed by the zinc trap assay. (115)(116)(117)(118) Electrochemical Sulfide ion specific electrode measures S 2form of sulfide which requires alkaline environment using Ag/Ag 2 S electrodes. (119,120) Polarographic real time measurement of H 2 S using a polarographic oxygen sensor as anode and platinum wire as cathode and alkaline K 3 Fe(CN) 6 as electrolyte. An H 2 S permeable membrane allows diffusion of H 2 S into the electrolyte solution reducing it. The electrolyte subsequently gets re-oxidized on the surface of the platinum electrode to produce a current proportional to H 2 S concentration. (121,122) Fluorescent sensors All fluorescent probes consist of a fluorescence signal transducer and the fluorescence modulator. The transducer is a suitable fluorescent moiety while the modulator is chosen based on the chemical nature of H 2 S and physiologically permissible reaction kinetics. Fluorescent moieties like rhodamine, BODIPY, dansyl, 7-hydroxy-4-methylcoumarin, naphthilamide, cyanine, etc, have been used as transducers. The modulators have been designed based on the selective reduction of nitro groups to amines and azide groups to amines by H 2 S, thiolysis, addition and cyclization reactions based on the nucleophilic nature of H 2 S and copper sulfide precipitation resulting from the affinity of H 2 S for copper. Others include selenium-based probes for reversible detection of H 2 S. Probes have been designed based on the selenide-selenoxide redox reaction of many selenoenzymes. These probes can monitor redox cycling between H 2 S and ROS.
Nanotechnology-based sensors Single walled carbon nanotube networks, gold nanoclusters, nanorods, nanocomposites of FAM DNA etched on the surface of silver nanoparticles have been employed for the detection of H 2 S. (124)(125)(126)(127) IUBMB Life. Author manuscript; available in PMC 2018 July 03.