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H2S and reactive sulfur signaling at the host-bacterial pathogen interface

Open AccessPublished:July 22, 2020DOI:https://doi.org/10.1074/jbc.REV120.011304
      Bacterial pathogens that cause invasive disease in the vertebrate host must adapt to host efforts to cripple their viability. Major host insults are reactive oxygen and reactive nitrogen species as well as cellular stress induced by antibiotics. Hydrogen sulfide (H2S) is emerging as an important player in cytoprotection against these stressors, which may well be attributed to downstream more oxidized sulfur species termed reactive sulfur species (RSS). In this review, we summarize recent work that suggests that H2S/RSS impacts bacterial survival in infected cells and animals. We discuss the mechanisms of biogenesis and clearance of RSS in the context of a bacterial H2S/RSS homeostasis model and the bacterial transcriptional regulatory proteins that act as “sensors” of cellular RSS that maintain H2S/RSS homeostasis. In addition, we cover fluorescence imaging– and MS–based approaches used to detect and quantify RSS in bacterial cells. Last, we discuss proteome persulfidation (S-sulfuration) as a potential mediator of H2S/RSS signaling in bacteria in the context of the writer-reader-eraser paradigm, and progress toward ascribing regulatory significance to this widespread post-translational modification.
      Infectious disease is a global and significant threat to human health. There is an increasingly urgent need to develop new antimicrobial strategies to combat these increasingly drug-resistant and life-threatening pathogens (
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      ) to limit bacterial growth. Pathogens, in turn, adapt by employing specialized transcriptional regulators, metallosensors, that sense metals and regulate the expression of genes encoding proteins that collectively maintain bioavailable metal in a range compatible with physiological needs (Fig. 1A, top) (
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      ). In an analogous fashion, bacteria encode specialized transcriptional regulators that sense oxidized or “reactive” sulfur species (RSS), derived from hydrogen sulfide (H2S) (
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      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
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      Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis.
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      FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.
      ,
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      • Trinidad J.C.
      • Skaar E.P.
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      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ). As cellular concentrations of RSS rise, RSS sensors turn on the expression of genes that encode enzymes that reduce cellular loads of H2S/RSS to avoid H2S toxicity and overpersulfidation of the metabolome and proteome (Fig. 1A, bottom). These RSS sensors, like metallosensors, control H2S/RSS homeostasis, allowing bacterial cells access to these molecules at low concentrations to meet physiological needs. In the infected host, H2S and RSS are derived from host cell metabolism, from commensal bacteria in polymicrobial communities, or from the pathogen itself. Recent studies that build on prior work (
      • Shatalin K.
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      H2S: a universal defense against antibiotics in bacteria.
      ) suggest that bacterial H2S biogenesis may well be a clinically important adaptive response during infections (
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      “On demand” redox buffering by H2S contributes to antibiotic resistance revealed by a bacteria-specific H2S donor.
      ).
      Figure thumbnail gr1
      Figure 1Set-point homeostasis models and speciation in bacteria. A, transition metal homeostasis (top) is orchestrated by a panel of metal-specific sensors that prevent metal starvation or toxicity by regulating the expression of proteins involved in the uptake, efflux, storage, or allocation of metals in cells (
      • Ma Z.
      • Jacobsen F.E.
      • Giedroc D.P.
      Coordination chemistry of bacterial metal transport and sensing.
      ,
      • Waldron K.J.
      • Rutherford J.C.
      • Ford D.
      • Robinson N.J.
      Metalloproteins and metal sensing.
      ,
      • Capdevila D.A.
      • Edmonds K.A.
      • Giedroc D.P.
      Metallochaperones and metalloregulation in bacteria.
      • Capdevila D.A.
      • Wang J.
      • Giedroc D.P.
      Bacterial strategies to maintain zinc metallostasis at the host-pathogen interface.
      ,
      • Jordan M.R.
      • Wang J.
      • Capdevila D.A.
      • Giedroc D.P.
      Multi-metal nutrient restriction and crosstalk in metallostasis systems in microbial pathogens.
      • Osman D.
      • Martini M.A.
      • Foster A.W.
      • Chen J.
      • Scott A.J.P.
      • Morton R.J.
      • Steed J.W.
      • Lurie-Luke E.
      • Huggins T.G.
      • Lawrence A.D.
      • Warren M.J.
      • Chivers P.T.
      • Robinson N.J.
      Bacterial sensors define intracellular free energies for correct enzyme metalation.
      ). Transcriptional response curves are shown for a pair of sensors that detect a specific metal (e.g. ZnII). These dual sensors collaboratively control metal bioavailability in a concentration range that is compatible with cellular physiology (gray box). H2S/RSS homeostasis (bottom) is achieved by a single RSS sensor that transcriptionally regulates the expression of enzymes involved in the biogenesis, clearance, transport, and assimilation of H2S/RSS (
      • Luebke J.L.
      • Shen J.
      • Bruce K.E.
      • Kehl-Fie T.E.
      • Peng H.
      • Skaar E.P.
      • Giedroc D.P.
      The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ,
      • Shimizu T.
      • Shen J.
      • Fang M.
      • Zhang Y.
      • Hori K.
      • Trinidad J.C.
      • Bauer C.E.
      • Giedroc D.P.
      • Masuda S.
      Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis.
      ,
      • Li H.
      • Li J.
      • Lu C.
      • Xia Y.
      • Xin Y.
      • Liu H.
      • Xun L.
      • Liu H.
      FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ). The transcriptional response of an RSS sensor detects a concentration range (gray box) that prevents cellular toxicity, while maintaining access to H2S/RSS that is physiologically beneficial at lower concentrations. B, metal speciation (top) of first row, late d-block transition metals is defined by the metallome, a descriptor of all oxidation states and coordination complexes in the cell, ranging from exchange-labile small-molecule metal complexes to protein cofactors (shown in cartoon form). Reactive sulfur speciation (bottom) is defined by all inorganic and organic small molecules that harbor sulfur atoms in oxidation states more positive than –2 (see key) (
      • Mishanina T.V.
      • Libiad M.
      • Banerjee R.
      Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways.
      ) and are collectively termed reactive sulfur species (RSS).
      A second feature that is common to metallostasis and H2S/RSS homeostasis, beyond the sensors themselves, is the concept of speciation (Fig. 1B). In metallostasis, speciation defines the metallome, or all coordination complexes, both small molecule and protein, and oxidation states of all transition metals in the cell (Fig. 1B, top). Metallosensors surveil the cytoplasm for some specific feature of the metallome (e.g. zinc in exchange-labile complexes) and alter gene expression upon metal binding. In H2S/RSS homeostasis, speciation is defined by the components of the RSS pool, which encompasses organic and inorganic molecules containing sulfur in oxidation states higher than H2S, many of which contain sulfur-bonded or “sulfane” sulfur (Fig. 1B, bottom) (
      • Mishanina T.V.
      • Libiad M.
      • Banerjee R.
      Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways.
      ). Analogous to a metallosensor, known RSS sensors specifically surveil the cytoplasm for a particular feature of the RSS pool, in this case sulfane sulfur (
      • Grossoehme N.
      • Kehl-Fie T.E.
      • Ma Z.
      • Adams K.W.
      • Cowart D.M.
      • Scott R.A.
      • Skaar E.P.
      • Giedroc D.P.
      Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.
      ,
      • Luebke J.L.
      • Shen J.
      • Bruce K.E.
      • Kehl-Fie T.E.
      • Peng H.
      • Skaar E.P.
      • Giedroc D.P.
      The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.
      ,
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ,
      • Shimizu T.
      • Shen J.
      • Fang M.
      • Zhang Y.
      • Hori K.
      • Trinidad J.C.
      • Bauer C.E.
      • Giedroc D.P.
      • Masuda S.
      Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis.
      ,
      • Li H.
      • Li J.
      • Lu C.
      • Xia Y.
      • Xin Y.
      • Liu H.
      • Xun L.
      • Liu H.
      FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.
      ,
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ).
      In this review, we summarize the biogenesis and clearance of H2S/RSS and the potential role these molecules play in bacterial infections. In addition, we discuss the molecular mechanisms of RSS sensors that maintain H2S/RSS homeostasis in bacteria. Elucidation of how H2S/RSS are leveraged in bacteria at the host-pathogen interface relies on the development of molecular tools to identify, detect, and quantify H2S and RSS as well as small-molecule probes to generate these species in vitro or in vivo. Last, we discuss recent efforts to detect and understand the regulatory significance of protein persulfidation (S-sulfuration) in bacteria.

      Hydrogen sulfide and reactive sulfur species in bacteria

      H2S is an electron-rich molecule historically well-known to drive photosynthesis (
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      ). In 2011, Nudler and co-workers (
      • Shatalin K.
      • Shatalina E.
      • Mironov A.
      • Nudler E.
      H2S: a universal defense against antibiotics in bacteria.
      ) reported that endogenously synthesized H2S or application of exogenous sulfide salts protected multiple bacterial pathogens against a broad array of mechanistically distinct antibiotics when grown in culture. This initial report, despite few insights into a possible mechanism, suggested that H2S might have beneficial properties in human disease–causing microorganisms and has thus inspired considerable research over the last 10 years. These bacteria endogenously synthesize H2S utilizing bacterial homologs of the mammalian reverse transsulfuration pathway via “side” reactions catalyzed by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) (
      • Chen X.
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      • Kruger W.D.
      Production of the neuromodulator H2S by cystathionine β-synthase via the condensation of cysteine and homocysteine.
      ,
      • Chiku T.
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      H2S biogenesis by human cystathionine γ-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia.
      ,
      • Singh S.
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      PLP-dependent H2S biogenesis.
      ,
      • Singh S.
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      Relative contributions of cystathionine β-synthase and γ-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions.
      ) or from cysteine catabolism to 3-mercaptopyruvate (3-MP) via cysteine aminotransferase (CAT) (
      • Miyamoto R.
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      • Yamaguchi S.
      • Ito S.
      Contribution of cysteine aminotransferase and mercaptopyruvate sulfurtransferase to hydrogen sulfide production in peripheral neurons.
      ) (Fig. 2A). 3-MP is then converted to pyruvate and H2S by 3-MP sulfurtransferase (3MST) (
      • Kimura Y.
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      3-Mercaptopyruvate sulfurtransferase produces potential redox regulators cysteine- and glutathione-persulfide (Cys-SSH and GSSH) together with signaling molecules H2S2, H2S3 and H2S.
      ,
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      Structure and kinetic analysis of H2S production by human mercaptopyruvate sulfurtransferase.
      ) via the intermediacy of a protein persulfide, E-SSH (Fig. 2A). Bacteria generally encode either 3MST or CBS/CSE, and it was recently demonstrated that L-cysteine desulfhydrases and cysteine desulfurases also contribute to H2S biogenesis in Escherichia coli (
      • Li K.
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      ). In addition, two groups recently reported the discovery of a glycyl radical enzyme from Bilophila wadsworthia that catalyzes C–S bond cleavage in the catabolism of tissue-abundant taurine and the analogous alcohol isethionate (2-hydroxyethanesulfonate) (
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      ). This reaction produces sulfite (SO32-), which is reduced to H2S by a dissimilatory sulfite reductase, thus defining a novel pathway for H2S production by gut microbiota.
      Figure thumbnail gr2
      Figure 2Endogenous production of H2S and cross-talk between H2S/RSS and ROS/RNS in bacteria. A, the reverse transsulfuration pathway synthesizes L-cysteine from L-homocysteine via the intermediacy of L-cystathionine, which is catabolized by CAT, whose product is utilized by 3MST to generate pyruvate and H2S (
      • Chen X.
      • Jhee K.H.
      • Kruger W.D.
      Production of the neuromodulator H2S by cystathionine β-synthase via the condensation of cysteine and homocysteine.
      • Chiku T.
      • Padovani D.
      • Zhu W.
      • Singh S.
      • Vitvitsky V.
      • Banerjee R.
      H2S biogenesis by human cystathionine γ-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia.
      ,
      • Singh S.
      • Banerjee R.
      PLP-dependent H2S biogenesis.
      ,
      • Singh S.
      • Padovani D.
      • Leslie R.A.
      • Chiku T.
      • Banerjee R.
      Relative contributions of cystathionine β-synthase and γ-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions.
      ,
      • Miyamoto R.
      • Otsuguro K.
      • Yamaguchi S.
      • Ito S.
      Contribution of cysteine aminotransferase and mercaptopyruvate sulfurtransferase to hydrogen sulfide production in peripheral neurons.
      ,
      • Kimura Y.
      • Koike S.
      • Shibuya N.
      • Lefer D.
      • Ogasawara Y.
      • Kimura H.
      3-Mercaptopyruvate sulfurtransferase produces potential redox regulators cysteine- and glutathione-persulfide (Cys-SSH and GSSH) together with signaling molecules H2S2, H2S3 and H2S.
      ,
      • Kimura Y.
      • Toyofuku Y.
      • Koike S.
      • Shibuya N.
      • Nagahara N.
      • Lefer D.
      • Ogasawara Y.
      • Kimura H.
      Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain.
      • Yadav P.K.
      • Yamada K.
      • Chiku T.
      • Koutmos M.
      • Banerjee R.
      Structure and kinetic analysis of H2S production by human mercaptopyruvate sulfurtransferase.
      ). L-Homocysteine is also the immediate precursor to L-methionine. Both CBS and CSE catalyze a number of additional reactions with alternative substrates (red arrows) that generate endogenous H2S (
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ), illustrated in the dashed box. CBS, cystathionine-β-synthase, CSE, cystathionine-γ-lyase; MAT, methionine adenosyltransferase; MS, methionine synthase; SAHH, SAH hydrolase. B, small-molecule cross-talk between H2S/RSS and RNS and ROS. C, direct reaction of HS with protein nitrosothiols or disulfides and sulfenic acids induced by RNS and ROS, respectively, all result in the formation of the thiol persulfide with release of HNO, protein thiol (RSH), and water, respectively. Reaction of HS with sulfenic acids is believed to protect these protein thiols from irreversible overoxidation, indicated by the block arrow (right).
      With a sulfur oxidation state of “–2”, H2S and organic thiols (e.g. cysteine or GSH (RSH)) are in their most reduced forms and can only function as cellular reductants (
      • Mishanina T.V.
      • Libiad M.
      • Banerjee R.
      Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways.
      ,
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ). RSS harbor higher sulfur oxidation states, ranging from “–1” to “+6” (Fig. 1B, bottom). The organic thiol persulfide (hydropersulfide, RSSH) is of particular interest because of its “Janus” character and can function as either a nucleophile when deprotonated (RSS) or an electrophile when protonated (RSSH). Due to a considerably lower pKa than the corresponding thiol, the anionic form predominates at physiological pH (
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ,
      • Cuevasanta E.
      • Lange M.
      • Bonanata J.
      • Coitiño E.L.
      • Ferrer-Sueta G.
      • Filipovic M.R.
      • Alvarez B.
      Reaction of hydrogen sulfide with disulfide and sulfenic acid to form the strongly nucleophilic persulfide.
      ,
      • Francoleon N.E.
      • Carrington S.J.
      • Fukuto J.M.
      The reaction of H2S with oxidized thiols: generation of persulfides and implications to H2S biology.
      ). Persulfides also have enhanced nucleophilicity compared with their corresponding thiolate because of the α-effect (
      • Edwards J.O.
      • Pearson R.G.
      The factors determining nucleophilic reactivities.
      ), which increases the reactivity of the terminal sulfur atom because of unpaired electrons in the adjacent atom.
      Persulfides readily react with oxidants such as hydrogen peroxide (H2O2) and peroxynitrite (
      • Cuevasanta E.
      • Lange M.
      • Bonanata J.
      • Coitiño E.L.
      • Ferrer-Sueta G.
      • Filipovic M.R.
      • Alvarez B.
      Reaction of hydrogen sulfide with disulfide and sulfenic acid to form the strongly nucleophilic persulfide.
      ,
      • Ida T.
      • Sawa T.
      • Ihara H.
      • Tsuchiya Y.
      • Watanabe Y.
      • Kumagai Y.
      • Suematsu M.
      • Motohashi H.
      • Fujii S.
      • Matsunaga T.
      • Yamamoto M.
      • Ono K.
      • Devarie-Baez N.O.
      • Xian M.
      • Fukuto J.M.
      • et al.
      Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling.
      ) and are superior one-electron reductants to thiols and H2S as reviewed elsewhere (
      • Benson S.W.
      Thermochemistry and kinetics of sulfur-containing molecules and radicals.
      ,
      • Everett S.A.
      • Wardman P.
      Perthiols as antioxidants: radical-scavenging and prooxidative mechanisms.
      ,
      • Koppenol W.H.
      • Bounds P.L.
      Signaling by sulfur-containing molecules: quantitative aspects.
      ). Their Janus character, enhanced nucleophilicity, and superior reducing capabilities make RSSH, along with organic polysulfides and their inorganic counterparts, potent antioxidants (Fig. 1B, bottom) (
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ,
      • Benchoam D.
      • Cuevasanta E.
      • Möller M.N.
      • Alvarez B.
      Hydrogen sulfide and persulfides oxidation by biologically relevant oxidizing species.
      ,
      • Ezerina D.
      • Takano Y.
      • Hanaoka K.
      • Urano Y.
      • Dick T.P.
      N-Acetyl cysteine functions as a fast-acting antioxidant by triggering intracellular H2S and sulfane sulfur production.
      ). These properties may well be responsible for many of the beneficial traits attributed to H2S, including protection against oxidative stress and antibiotics in the infected host (
      • Shatalin K.
      • Shatalina E.
      • Mironov A.
      • Nudler E.
      H2S: a universal defense against antibiotics in bacteria.
      ,
      • Shukla P.
      • Khodade V.S.
      • SharathChandra M.
      • Chauhan P.
      • Mishra S.
      • Siddaramappa S.
      • Pradeep B.E.
      • Singh A.
      • Chakrapani H.
      “On demand” redox buffering by H2S contributes to antibiotic resistance revealed by a bacteria-specific H2S donor.
      ,
      • Ida T.
      • Sawa T.
      • Ihara H.
      • Tsuchiya Y.
      • Watanabe Y.
      • Kumagai Y.
      • Suematsu M.
      • Motohashi H.
      • Fujii S.
      • Matsunaga T.
      • Yamamoto M.
      • Ono K.
      • Devarie-Baez N.O.
      • Xian M.
      • Fukuto J.M.
      • et al.
      Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling.
      ).
      Recent work by several groups reveals significant physiological overlap or cross-talk between H2S/RSS and reactive oxygen (ROS) and reactive nitrogen (RNS) species. Oxidation of RSS results in the production of inorganic sulfur-containing molecules sulfite, thiosulfate, and sulfate (Fig. 2B) (
      • Benchoam D.
      • Cuevasanta E.
      • Möller M.N.
      • Alvarez B.
      Hydrogen sulfide and persulfides oxidation by biologically relevant oxidizing species.
      ). ROS can also drive the formation of low-molecular weight (LMW) thiol disulfides (RSSR) and sulfenic acids (RSOH), a major physiological marker of H2O2 reactivity, which reacts with HS to form organic RSS (Fig. 2B). H2S and nitric oxide (NO·) intersect via nitroxyl (HNO), and incubation of bacterial cells with a nitroxyl donor, Angeli's salt, results in an increase in cellular levels of RSS in Staphylococcus aureus (
      • Peng H.
      • Shen J.
      • Edmonds K.A.
      • Luebke J.L.
      • Hickey A.K.
      • Palmer L.D.
      • Chang F.J.
      • Bruce K.A.
      • Kehl-Fie T.E.
      • Skaar E.P.
      • Giedroc D.P.
      Sulfide homeostasis and nitroxyl intersect via formation of reactive sulfur species in Staphylococcus aureus.
      ) possibly via thionitrous acid (HSNO) or nitrosopersulfide (SSNO) formation (Fig. 2B) (
      • Bailey T.S.
      • Henthorn H.A.
      • Pluth M.D.
      The intersection of NO and H2S: persulfides generate NO from nitrite through polysulfide formation.
      ,
      • Cortese-Krott M.M.
      • Kuhnle G.G.C.
      • Dyson A.
      • Fernandez B.O.
      • Grman M.
      • DuMond J.F.
      • Barrow M.P.
      • McLeod G.
      • Nakagawa H.
      • Ondrias K.
      • Nagy P.
      • King S.B.
      • Saavedra J.E.
      • Keefer L.K.
      • Singer M.
      • et al.
      Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl.
      ,
      • Ivanovic-Burmazovic I.
      • Filipovic M.R.
      Saying NO to H2S: a Story of HNO, HSNO, and SSNO.
      ). In addition, polysulfides can by synthesized from incubation of RSH with sodium nitrite (NO2-) to form organic nitrosothiols (RSNO), which readily react with HS at acidic pH to form a mixture of RS–Sn–SR, consistent with proposed H2S/NO· cross-talk (
      • Zhang T.
      • Ono K.
      • Tsutsuki H.
      • Ihara H.
      • Islam W.
      • Akaike T.
      • Sawa T.
      Enhanced cellular polysulfides negatively regulate TLR4 signaling and mitigate lethal endotoxin shock.
      ). Last, protein persulfidation (S-sulfuration) is now a widely recognized post-translational modification (PTM) believed to function in H2S signaling alongside, and possibly interconverting with, other thiol modifications, including S-thiolation (RSSR′), S-nitrosation (RSNO), or oxidation to sulfenic, sulfinic (RSO2H), and sulfonic acids (RSO3H) (Fig. 2C) (
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ,
      • Mustafa A.K.
      • Gadalla M.M.
      • Sen N.
      • Kim S.
      • Mu W.
      • Gazi S.K.
      • Barrow R.K.
      • Yang G.
      • Wang R.
      • Snyder S.H.
      H2S signals through protein S-sulfhydration.
      ,
      • Paul B.D.
      • Snyder S.H.
      H2S signalling through protein sulfhydration and beyond.
      ,
      • Paul B.D.
      • Snyder S.H.
      Protein sulfhydration.
      ). It is important to note that the chemistry presented here between H2S/RSS and ROS/RNS can potentially occur on both small-molecule and protein thiols (Fig. 2, B and C). Furthermore, the onslaught of host-generated ROS and RNS at sites of infection suggests this chemical cross-talk may be biologically relevant in the infected host (
      • Luebke J.L.
      • Giedroc D.P.
      Cysteine sulfur chemistry in transcriptional regulators at the host-bacterial pathogen interface.
      ,
      • Tharmalingam S.
      • Alhasawi A.
      • Appanna V.P.
      • Lemire J.
      • Appanna V.D.
      Reactive nitrogen species (RNS)-resistant microbes: adaptation and medical implications.
      ,
      • Winterbourn C.C.
      • Kettle A.J.
      • Hampton M.B.
      Reactive oxygen species and neutrophil function.
      ,
      • Yang Y.
      • Bazhin A.V.
      • Werner J.
      • Karakhanova S.
      Reactive oxygen species in the immune system.
      ).

      Physiological conditions for the production, regulation, and signaling of H2S/RSS in bacteria

      H2S and the gut microbiome

      The gut microbiome is a complex, nutrient-rich environment and host to well over 100 bacterial species (
      • Ley R.E.
      • Peterson D.A.
      • Gordon J.I.
      Ecological and evolutionary forces shaping microbial diversity in the human intestine.
      ,
      • Qin J.
      • Li R.
      • Raes J.
      • Arumugam M.
      • Burgdorf K.S.
      • Manichanh C.
      • Nielsen T.
      • Pons N.
      • Levenez F.
      • Yamada T.
      • Mende D.R.
      • Li J.
      • Xu J.
      • Li S.
      • Li D.
      • et al.
      MetaHIT Consortium
      A human gut microbial gene catalogue established by metagenomic sequencing.
      ). The microorganisms that inhabit this niche are a significant endogenous source of sulfur-containing compounds and H2S, the latter estimated to range from ∼0.2 to 2.4 mm (Fig. 3A) (
      • Macfarlane G.T.
      • Gibson G.R.
      • Cummings J.H.
      Comparison of fermentation reactions in different regions of the human colon.
      ). Methionine catabolism to cysteine via the reverse transsulfuration pathway (Fig. 2A) and the catabolism of organic sulfonates, notably taurine, are known to be catalyzed by gut microbiota (
      • Peck S.C.
      • Denger K.
      • Burrichter A.
      • Irwin S.M.
      • Balskus E.P.
      • Schleheck D.
      A glycyl radical enzyme enables hydrogen sulfide production by the human intestinal bacterium Bilophila wadsworthia.
      ,
      • Xing M.
      • Wei Y.
      • Zhou Y.
      • Zhang J.
      • Lin L.
      • Hu Y.
      • Hua G.
      • A N.N.U.
      • Liu D.
      • Wang F.
      • Guo C.
      • Tong Y.
      • Li M.
      • Liu Y.
      • Ang E.L.
      • et al.
      Radical-mediated C-S bond cleavage in C2 sulfonate degradation by anaerobic bacteria.
      ,
      • Riedijk M.A.
      • Stoll B.
      • Chacko S.
      • Schierbeek H.
      • Sunehag A.L.
      • van Goudoever J.B.
      • Burrin D.G.
      Methionine transmethylation and transsulfuration in the piglet gastrointestinal tract.
      ,
      • Shoveller A.K.
      • Brunton J.A.
      • House J.D.
      • Pencharz P.B.
      • Ball R.O.
      Dietary cysteine reduces the methionine requirement by an equal proportion in both parenterally and enterally fed piglets.
      ,
      • Stegink L.D.
      • Besten L.D.
      Synthesis of cysteine from methionine in normal adult subjects: effect of route of alimentation.
      ,
      • Zlotkin S.H.
      • Bryan M.H.
      • Anderson G.H.
      Cysteine supplementation to cysteine-free intravenous feeding regimens in newborn infants.
      ,
      • Barton L.L.
      • Ritz N.L.
      • Fauque G.D.
      • Lin H.C.
      Sulfur cycling and the intestinal microbiome.
      ). This niche is also home to sulfate-reducing bacteria that are responsible for significant production of H2S (
      • Gibson G.R.
      • Macfarlane G.T.
      • Cummings J.H.
      Occurrence of sulphate-reducing bacteria in human faeces and the relationship of dissimilatory sulphate reduction to methanogenesis in the large gut.
      ,
      • Macfarlane G.T.
      • Cummings J.H.
      • Macfarlane S.
      Sulphate-reducing bacteria and the human large intestine.
      ), in addition to the reduction of tetrathionate and thiosulfate to H2S that also occurs in the gut (
      • Barrett E.L.
      • Clark M.A.
      Tetrathionate reduction and production of hydrogen sulfide from thiosulfate.
      ). H2S produced by the gut microbiota is oxidized by gastrointestinal epithelial cells to thiosulfate and tetrathionate (
      • Libiad M.
      • Yadav P.K.
      • Vitvitsky V.
      • Martinov M.
      • Banerjee R.
      Organization of the human mitochondrial hydrogen sulfide oxidation pathway.
      ,
      • Libiad M.
      • Vitvitsky V.
      • Bostelaar T.
      • Bak D.W.
      • Lee H.J.
      • Sakamoto N.
      • Fearon E.
      • Lyssiotis C.A.
      • Weerapana E.
      • Banerjee R.
      Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.
      ); these molecules in turn are utilized by the microbiota as electron acceptors, resulting in a symbiotic relationship derived from interconversion of sulfur species (Fig. 3A) (
      • Barton L.L.
      • Ritz N.L.
      • Fauque G.D.
      • Lin H.C.
      Sulfur cycling and the intestinal microbiome.
      ). Perturbation of this symbiotic relationship results in the accumulation of H2S now linked to several gut-derived diseases (
      • Pitcher M.C.
      • Cummings J.H.
      Hydrogen sulphide: a bacterial toxin in ulcerative colitis?.
      ,
      • Attene-Ramos M.S.
      • Wagner E.D.
      • Plewa M.J.
      • Gaskins H.R.
      Evidence that hydrogen sulfide is a genotoxic agent.
      ,
      • Xu G.Y.
      • Winston J.H.
      • Shenoy M.
      • Zhou S.
      • Chen J.D.
      • Pasricha P.J.
      The endogenous hydrogen sulfide producing enzyme cystathionine-β synthase contributes to visceral hypersensitivity in a rat model of irritable bowel syndrome.
      ). Interestingly, gut inflammation caused by the pathogen Salmonella enterica serovar Typhimurium results in increased production of tetrathionate from the oxidation of thiosulfate by gut inflammation–derived ROS, which this bacterium uses as an alternate electron acceptor, thus providing a growth advantage in this niche (
      • Winter S.E.
      • Thiennimitr P.
      • Winter M.G.
      • Butler B.P.
      • Huseby D.L.
      • Crawford R.W.
      • Russell J.M.
      • Bevins C.L.
      • Adams L.G.
      • Tsolis R.M.
      • Roth J.R.
      • Bäumler A.J.
      Gut inflammation provides a respiratory electron acceptor for Salmonella.
      ).
      Figure thumbnail gr3
      Figure 3H2S and RSS at the host-pathogen interface. A, common sulfur sources in the gastrointestinal tract derived from host epithelial cells and dietary sulfur metabolized by the gut microbiota. B, endogenous production of H2S via CBS, CSE, and/or 3MST (see A) or other enzymatic processes and more oxidized RSS are cytoprotective against myriad stressors of innate immune response or by antibiotics. See section on physiological conditions for the production, regulation, and signaling of H2S/RSS in bacteria for details. PspE, single-domain sulfurtransferase in E. coli; Q/QH2, quinone; SQR, sulfide:quinone oxidoreductase.

      H2S at the host-pathogen interface

      There are two major physiological conditions within the infected host where H2S and downstream RSS may enhance bacterial survival in what is otherwise a hostile microenvironment. These are (i) resistance against myriad oxidative stressors and antibiotic challenge and (ii) H2S/RSS-dependent regulation of biofilm dynamics. The host immune system produces diverse ROS, including O2¯ (
      • Babior B.M.
      • Kipnes R.S.
      • Curnutte J.T.
      Biological defense mechanisms: the production by leukocytes of superoxide, a potential bactericidal agent.
      ,
      • Goldstein I.M.
      • Roos D.
      • Kaplan H.B.
      • Weissmann G.
      Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis.
      ,
      • Johnston Jr., R.B.
      • Keele Jr., B.B.
      • Misra H.P.
      • Lehmeyer J.E.
      • Webb L.S.
      • Baehner R.L.
      • RaJagopalan K.V.
      The role of superoxide anion generation in phagocytic bactericidal activity: studies with normal and chronic granulomatous disease leukocytes.
      ), hydrogen peroxide (H2O2) (
      • Root R.K.
      • Metcalf J.
      • Oshino N.
      • Chance B.
      H2O2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors.
      ), and hydroxy radical (OH·) (
      • Tauber A.I.
      • Babior B.M.
      Evidence for hydroxyl radical production by human neutrophils.
      ) to combat bacterial infections (
      • Mundi H.
      • Björkstén B.
      • Svanborg C.
      • Ohman L.
      • Dahlgren C.
      Extracellular release of reactive oxygen species from human neutrophils upon interaction with Escherichia coli strains causing renal scarring.
      ). Antibiotics are also thought to induce generalized oxidative stress (
      • Albesa I.
      • Becerra M.C.
      • Battán P.C.
      • Páez P.L.
      Oxidative stress involved in the antibacterial action of different antibiotics.
      ,
      • Kohanski M.A.
      • Dwyer D.J.
      • Hayete B.
      • Lawrence C.A.
      • Collins J.J.
      A common mechanism of cellular death induced by bactericidal antibiotics.
      ,
      • Dwyer D.J.
      • Kohanski M.A.
      • Hayete B.
      • Collins J.J.
      Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli.
      ,
      • Wang X.
      • Zhao X.
      Contribution of oxidative damage to antimicrobial lethality.
      ), although this has been widely debated in recent years (
      • Keren I.
      • Wu Y.
      • Inocencio J.
      • Mulcahy L.R.
      • Lewis K.
      Killing by bactericidal antibiotics does not depend on reactive oxygen species.
      ,
      • Liu Y.
      • Imlay J.A.
      Cell death from antibiotics without the involvement of reactive oxygen species.
      ). The first documentation that H2S impacted antibiotic resistance in E. coli was published 47 years ago (
      • Darland G.
      • Davis B.R.
      Biochemical and serological characterization of hydrogen sulfide-positive variants of Escherichia coli.
      ). Renewed interest came in 2011 when it was demonstrated that bacterially derived H2S conferred resistance to a broad range of antibiotics in several bacterial pathogens (
      • Shatalin K.
      • Shatalina E.
      • Mironov A.
      • Nudler E.
      H2S: a universal defense against antibiotics in bacteria.
      ). Bacterially derived NO· has also been found to provide protection against antibiotics by the same group (
      • Gusarov I.
      • Shatalin K.
      • Starodubtseva M.
      • Nudler E.
      Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics.
      ) perhaps because of H2S/NO· cross-talk, which has only been investigated in recent years (Fig. 2B) (
      • Peng H.
      • Shen J.
      • Edmonds K.A.
      • Luebke J.L.
      • Hickey A.K.
      • Palmer L.D.
      • Chang F.J.
      • Bruce K.A.
      • Kehl-Fie T.E.
      • Skaar E.P.
      • Giedroc D.P.
      Sulfide homeostasis and nitroxyl intersect via formation of reactive sulfur species in Staphylococcus aureus.
      ,
      • Bailey T.S.
      • Henthorn H.A.
      • Pluth M.D.
      The intersection of NO and H2S: persulfides generate NO from nitrite through polysulfide formation.
      ,
      • Cortese-Krott M.M.
      • Kuhnle G.G.C.
      • Dyson A.
      • Fernandez B.O.
      • Grman M.
      • DuMond J.F.
      • Barrow M.P.
      • McLeod G.
      • Nakagawa H.
      • Ondrias K.
      • Nagy P.
      • King S.B.
      • Saavedra J.E.
      • Keefer L.K.
      • Singer M.
      • et al.
      Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl.
      ,
      • Ivanovic-Burmazovic I.
      • Filipovic M.R.
      Saying NO to H2S: a Story of HNO, HSNO, and SSNO.
      ). It was not until 2014 that RSS were shown to function as antioxidants in mammalian cells (
      • Ida T.
      • Sawa T.
      • Ihara H.
      • Tsuchiya Y.
      • Watanabe Y.
      • Kumagai Y.
      • Suematsu M.
      • Motohashi H.
      • Fujii S.
      • Matsunaga T.
      • Yamamoto M.
      • Ono K.
      • Devarie-Baez N.O.
      • Xian M.
      • Fukuto J.M.
      • et al.
      Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling.
      ), which may partly explain H2S-enhanced bacterial resistance to antibiotics (
      • Shatalin K.
      • Shatalina E.
      • Mironov A.
      • Nudler E.
      H2S: a universal defense against antibiotics in bacteria.
      ). RSS have been detected in a number of bacterial pathogens (
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ,
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ,
      • Peng H.
      • Shen J.
      • Edmonds K.A.
      • Luebke J.L.
      • Hickey A.K.
      • Palmer L.D.
      • Chang F.J.
      • Bruce K.A.
      • Kehl-Fie T.E.
      • Skaar E.P.
      • Giedroc D.P.
      Sulfide homeostasis and nitroxyl intersect via formation of reactive sulfur species in Staphylococcus aureus.
      ,
      • Peng H.
      • Zhang Y.
      • Palmer L.D.
      • Kehl-Fie T.E.
      • Skaar E.P.
      • Trinidad J.C.
      • Giedroc D.P.
      Hydrogen sulfide and reactive sulfur species impact proteome S-sulfhydration and global virulence regulation in Staphylococcus aureus.
      ) and are the subject of ongoing work to better understand the role of RSS in the bacterial response to the host immune system.
      Recent work has investigated the mechanism by which H2S/RSS might confer antibiotic resistance and protection against ROS and sulfide toxicity (Fig. 3B). In E. coli, increased H2S results in a respiratory flux switch from that of the primary cytochrome bo oxidase to the alternate cytochrome bd oxidase, a copper-free enzyme that is far less susceptible to inhibition by H2S (
      • Shukla P.
      • Khodade V.S.
      • SharathChandra M.
      • Chauhan P.
      • Mishra S.
      • Siddaramappa S.
      • Pradeep B.E.
      • Singh A.
      • Chakrapani H.
      “On demand” redox buffering by H2S contributes to antibiotic resistance revealed by a bacteria-specific H2S donor.
      ,
      • Korshunov S.
      • Imlay K.R.
      • Imlay J.A.
      The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide.
      ). Although cytochrome bd oxidase does not pump protons, it still enables aerobic metabolism and robust growth. This respiratory switch in response to H2S may well occur in several other bacterial pathogens, including Acinetobacter baumannii (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ) and Mycobacterium tuberculosis; in the latter case, low levels of H2S enhance the respiration, energy production, and survival of M. tuberculosis in infected mice (
      • Saini V.
      • Chinta K.C.
      • Reddy V.P.
      • Glasgow J.N.
      • Stein A.
      • Lamprecht D.A.
      • Rahman M.A.
      • Mackenzie J.S.
      • Truebody B.E.
      • Adamson J.H.
      • Kunota T.T.R.
      • Bailey S.M.
      • Moellering D.R.
      • Lancaster Jr., J.R.
      • Steyn A.J.C.
      Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration, growth and pathogenesis.
      ). Further studies are required to establish the generality of this adaptive response across a wider range of organisms. Others have posited that H2S-mediated cytoprotection occurs via sequestration of the prooxidant free Fe(II) (
      • Mironov A.
      • Seregina T.
      • Nagornykh M.
      • Luhachack L.G.
      • Korolkova N.
      • Lopes L.E.
      • Kotova V.
      • Zavilgelsky G.
      • Shakulov R.
      • Shatalin K.
      • Nudler E.
      Mechanism of H2S-mediated protection against oxidative stress in Escherichia coli.
      ,
      • Ooi X.J.
      • Tan K.S.
      Reduced glutathione mediates resistance to H2S toxicity in oral Streptococci.
      ); however, this remains incompletely understood.
      A number of recent reports have described a potential role of H2S at the host-pathogen interface beyond protection against antibiotic and oxidative stress (
      • Toliver-Kinsky T.
      • Cui W.
      • Törö G.
      • Lee S.-J.
      • Shatalin K.
      • Nudler E.
      • Szabo C.
      H2S, a bacterial defense mechanism against the host immune response.
      ,
      • Luhachack L.
      • Rasouly A.
      • Shamovsky I.
      • Nudler E.
      Transcription factor YcjW controls the emergency H2S production in E. coli.
      ,
      • Saini V.
      • Chinta K.C.
      • Reddy V.P.
      • Glasgow J.N.
      • Stein A.
      • Lamprecht D.A.
      • Rahman M.A.
      • Mackenzie J.S.
      • Truebody B.E.
      • Adamson J.H.
      • Kunota T.T.R.
      • Bailey S.M.
      • Moellering D.R.
      • Lancaster Jr., J.R.
      • Steyn A.J.C.
      Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration, growth and pathogenesis.
      ,
      • Gobert A.P.
      • Latour Y.L.
      • Asim M.
      • Finley J.L.
      • Verriere T.G.
      • Barry D.P.
      • Milne G.L.
      • Luis P.B.
      • Schneider C.
      • Rivera E.S.
      • Lindsey-Rose K.
      • Schey K.L.
      • Delgado A.G.
      • Sierra J.C.
      • Piazuelo M.B.
      • et al.
      Bacterial pathogens hijack the innate immune response by activation of the reverse transsulfuration pathway.
      ). In infected macrophages and in mice, Helicobacter pylori was found to induce the expression of the host transsulfuration pathway enzyme CSE (Fig. 2A), resulting in increased cystathionine production that enhances H. pylori survival in these models (
      • Gobert A.P.
      • Latour Y.L.
      • Asim M.
      • Finley J.L.
      • Verriere T.G.
      • Barry D.P.
      • Milne G.L.
      • Luis P.B.
      • Schneider C.
      • Rivera E.S.
      • Lindsey-Rose K.
      • Schey K.L.
      • Delgado A.G.
      • Sierra J.C.
      • Piazuelo M.B.
      • et al.
      Bacterial pathogens hijack the innate immune response by activation of the reverse transsulfuration pathway.
      ). Any connection of cystathionine to host or bacterially derived H2S was not elucidated in this work. S. aureus and E. coli strains lacking enzymes involved in H2S biogenesis are more readily cleared in infected macrophages and are less resistant to leukocyte-mediated killing in a burn-infection model (
      • Toliver-Kinsky T.
      • Cui W.
      • Törö G.
      • Lee S.-J.
      • Shatalin K.
      • Nudler E.
      • Szabo C.
      H2S, a bacterial defense mechanism against the host immune response.
      ). In addition, E. coli strains lacking the H2S-generating enzyme 3MST, when challenged with antibiotics, give rise to a suppressor mutation that recovers H2S biogenesis via up-regulation of the single-domain sulfurtransferase PspE (
      • Luhachack L.
      • Rasouly A.
      • Shamovsky I.
      • Nudler E.
      Transcription factor YcjW controls the emergency H2S production in E. coli.
      ). Together, these studies suggest that H2S biogenesis reduces the efficacy of antibiotics and that up-regulation of H2S may be a clinically important adaptive response during infections. These studies support the proposal that H2S functions as an infection-relevant antioxidant or pro-antioxidant, in the latter case, as a precursor to oxidized RSS (
      • Ida T.
      • Sawa T.
      • Ihara H.
      • Tsuchiya Y.
      • Watanabe Y.
      • Kumagai Y.
      • Suematsu M.
      • Motohashi H.
      • Fujii S.
      • Matsunaga T.
      • Yamamoto M.
      • Ono K.
      • Devarie-Baez N.O.
      • Xian M.
      • Fukuto J.M.
      • et al.
      Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling.
      ).
      Whereas H2S/RSS-dependent regulation of biofilm dynamics remains largely unknown, recent studies suggest a potential connection. Biofilms are often polymicrobial communities that assemble on both abiotic (e.g. catheters and implants) and biotic (e.g. cells and cell debris) surfaces while conferring increased resistance to antibiotics (
      • Davies D.
      Understanding biofilm resistance to antibacterial agents.
      ,
      • Prax M.
      • Bertram R.
      Metabolic aspects of bacterial persisters.
      ,
      • Sonderholm M.
      • Bjarnsholt T.
      • Alhede M.
      • Kolpen M.
      • Jensen P.O.
      • Kuhl M.
      • Kragh K.N.
      The consequences of being in an infectious biofilm: microenvironmental conditions governing antibiotic tolerance.
      ). Cells near the base of biofilm structures are often nutrient-poor, and some reside at an oxic/anoxic boundary. Low-O2 (hypoxic) conditions can also result from increased O2 consumption by host immune cells to produce superoxide anion (O2¯) (
      • Babior B.M.
      • Curnutte J.T.
      • McMurrich B.J.
      The particulate superoxide-forming system from human neutrophils: properties of the system and further evidence supporting its participation in the respiratory burst.
      ,
      • Kolpen M.
      • Hansen C.R.
      • Bjarnsholt T.
      • Moser C.
      • Christensen L.D.
      • van Gennip M.
      • Ciofu O.
      • Mandsberg L.
      • Kharazmi A.
      • Döring G.
      • Givskov M.
      • Høiby N.
      • Jensen P.Ø.
      Polymorphonuclear leucocytes consume oxygen in sputum from chronic Pseudomonas aeruginosa pneumonia in cystic fibrosis.
      ). In these low-O2 regions, bacteria respire via reduction of nitrate (NO3-), producing NO· on pathway to nitrous oxide (N2O) and dinitrogen (N2) (
      • Line L.
      • Alhede M.
      • Kolpen M.
      • Kühl M.
      • Ciofu O.
      • Bjarnsholt T.
      • Moser C.
      • Toyofuku M.
      • Nomura N.
      • Høiby N.
      • Jensen P.Ã.
      Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum.
      ). These nitrogen-containing species have been reported to lead to biofilm dissemination of S. aureus (
      • Schlag S.
      • Nerz C.
      • Birkenstock T.A.
      • Altenberend F.
      • Götz F.
      Inhibition of staphylococcal biofilm formation by nitrite.
      ) and P. aeruginosa (
      • Barraud N.
      • Hassett D.J.
      • Hwang S.-H.
      • Rice S.A.
      • Kjelleberg S.
      • Webb J.S.
      Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa.
      ,
      • Barraud N.
      • Schleheck D.
      • Klebensberger J.
      • Webb J.S.
      • Hassett D.J.
      • Rice S.A.
      • Kjelleberg S.
      Nitric oxide signaling in Pseudomonas aeruginosa biofilms mediates phosphodiesterase activity, decreased cyclic di-GMP levels, and enhanced dispersal.
      ), consistent with an impact on biofilm dynamics.
      Redox homeostasis is also implicated in proper biofilm formation in P. aeruginosa (
      • Klare W.
      • Das T.
      • Ibugo A.
      • Buckle E.
      • Manefield M.
      • Manos J.
      Glutathione-disrupted biofilms of clinical Pseudomonas aeruginosa strains exhibit an enhanced antibiotic effect and a novel biofilm transcriptome.
      ), whereas cysteine and GSH-deficient uropathogenic E. coli exhibit dysregulated biofilm formation that is restored upon the addition of exogenous thiols (
      • Hufnagel D.A.
      • Price J.E.
      • Stephenson R.E.
      • Kelley J.
      • Benoit M.F.
      • Chapman M.R.
      Thiol starvation induces redox-mediated dysregulation of Escherichia coli biofilm components.
      ). Although the connection between biofilm regulation and H2S/RSS homeostasis is largely speculation at this point, H2S has been detected in cystic fibrosis sputum, a complex biofilm (
      • Cowley E.S.
      • Kopf S.H.
      • LaRiviere A.
      • Ziebis W.
      • Newman D.K.
      Pediatric cystic fibrosis sputum can be chemically dynamic, anoxic, and extremely reduced due to hydrogen sulfide formation.
      ), and H2S has been found to promote formation of biofilms by intestinal microbiota while reducing the proliferation of planktonic bacterial cells (
      • Motta J.-P.
      • Flannigan K.L.
      • Agbor T.A.
      • Beatty J.K.
      • Blackler R.W.
      • Workentine M.L.
      • Da Silva G.J.
      • Wang R.
      • Buret A.G.
      • Wallace J.L.
      Hydrogen sulfide protects from colitis and restores intestinal microbiota biofilm and mucus production.
      ). We recently characterized the biofilm growth–associated repressor, BigR, in A. baumannii as an RSS sensor (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ,
      • Capdevila D.A.
      • Walsh B.J.C.
      • Zhang Y.
      • Dietrich C.
      • Gonzalez-Gutierrez G.
      • Giedroc D.P.
      Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator.
      ), as previously characterized in plant pathogens (
      • Barbosa R.L.
      • Benedetti C.E.
      BigR, a transcriptional repressor from plant-associated bacteria, regulates an operon implicated in biofilm growth.
      ,
      • Guimarães B.G.
      • Barbosa R.L.
      • Soprano A.S.
      • Campos B.M.
      • de Souza T.A.
      • Tonoli C.C.C.
      • Leme A.F.P.
      • Murakami M.T.
      • Benedetti C.E.
      Plant pathogenic bacteria utilize biofilm growth-associated repressor (BigR), a novel winged-helix redox switch, to control hydrogen sulfide detoxification under hypoxia.
      ,
      • de Lira N.P.V.
      • Pauletti B.A.
      • Marques A.C.
      • Perez C.A.
      • Caserta R.
      • de Souza A.A.
      • Vercesi A.E.
      • Paes Leme A.F.
      • Benedetti C.E.
      BigR is a sulfide sensor that regulates a sulfur transferase/dioxygenase required for aerobic respiration of plant bacteria under sulfide stress.
      ). That work also identified two transcriptional regulators in A. baumannii known or projected to be involved in biofilm regulation that were characterized by significantly increased protein persulfidation mediated by exogenous sulfide (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ). Whereas these studies suggest that H2S/RSS homeostasis impacts biofilm dynamics, more studies are needed to better understand this connection at a mechanistic level.

      Biogenesis and clearance of organic RSS in bacteria

      The endogenous production of H2S in bacteria suggests that more oxidized forms of sulfur may be present in cells and formed via enzymatic and possibly nonenzymatic mechanisms (Fig. 4, A–C). The extent to which these pathways, particularly nonenzymatic routes, contribute to RSS pools in bacteria is not known and may well differ among organisms. Emerging evidence in mammalian systems demonstrates the role of ferric (FeIII)-heme in the oxidation of H2S, which reduces the FeIII to FeII and forms the one-electron oxidized radical HS· upon dissociation (Fig. 4A) (
      • Vitvitsky V.
      • Yadav P.K.
      • Kurthen A.
      • Banerjee R.
      Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides.
      ,
      • Bostelaar T.
      • Vitvitsky V.
      • Kumutima J.
      • Lewis B.E.
      • Yadav P.K.
      • Brunold T.C.
      • Filipovic M.
      • Lehnert N.
      • Stemmler T.L.
      • Banerjee R.
      Hydrogen sulfide oxidation by myoglobin.
      ,
      • Ruetz M.
      • Kumutima J.
      • Lewis B.E.
      • Filipovic M.R.
      • Lehnert N.
      • Stemmler T.L.
      • Banerjee R.
      A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin.
      ,
      • Vitvitsky V.
      • Miljkovic J.L.
      • Bostelaar T.
      • Adhikari B.
      • Yadav P.K.
      • Steiger A.K.
      • Torregrossa R.
      • Pluth M.D.
      • Whiteman M.
      • Banerjee R.
      • Filipovic M.R.
      Cytochrome c reduction by H2S potentiates sulfide signaling.
      ,
      • Nelp M.T.
      • Zheng V.
      • Davis K.M.
      • Stiefel K.J.E.
      • Groves J.T.
      Potent activation of indoleamine 2,3-dioxygenase by polysulfides.
      ). Recent work reveals that this mechanism is used to reactivate enzymes requiring a catalytically active ferrous heme from the inactive ferric state, formed during turnover (
      • Nelp M.T.
      • Zheng V.
      • Davis K.M.
      • Stiefel K.J.E.
      • Groves J.T.
      Potent activation of indoleamine 2,3-dioxygenase by polysulfides.
      ). Additionally, HS· can be formed by the reaction of H2S with superoxide radical anion O2¯ or with cysteine-coordinated Zn(II) sites in proteins (
      • Lange M.
      • Ok K.
      • Shimberg G.D.
      • Bursac B.
      • Markó L.
      • Ivanović-Burmazović I.
      • Michel S.L.J.
      • Filipovic M.R.
      Direct zinc finger protein persulfidation by H2S is facilitated by Zn(2).
      ). FeIII-heme has also been shown to result in formation of thiosulfate and hydropolysulfide species in mammalian systems (Fig. 4A) (
      • Vitvitsky V.
      • Yadav P.K.
      • Kurthen A.
      • Banerjee R.
      Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides.
      ,
      • Bostelaar T.
      • Vitvitsky V.
      • Kumutima J.
      • Lewis B.E.
      • Yadav P.K.
      • Brunold T.C.
      • Filipovic M.
      • Lehnert N.
      • Stemmler T.L.
      • Banerjee R.
      Hydrogen sulfide oxidation by myoglobin.
      ). Formation of organic thiyl radical, RS·, and related organic polysulfide species may occur via similar chemistry, supported by recent work using an LMW thiol for the reactivation of a catalytically active ferrous heme (Fig. 4B) (
      • Nelp M.T.
      • Zheng V.
      • Davis K.M.
      • Stiefel K.J.E.
      • Groves J.T.
      Potent activation of indoleamine 2,3-dioxygenase by polysulfides.
      ). The extent to which heme-based biogenesis of RSS occurs in bacteria is not yet known.
      Figure thumbnail gr4
      Figure 4Biogenesis and clearance of organic and inorganic RSS in bacteria. A and B, biogenesis of inorganic (A) and organic (B) RSS via enzymatic or nonenzymatic processes (
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ,
      • Benchoam D.
      • Cuevasanta E.
      • Möller M.N.
      • Alvarez B.
      Hydrogen sulfide and persulfides oxidation by biologically relevant oxidizing species.
      ). In A, the biogenesis of thiosulfate shown is a schematic rendering only. C, transsulfuration reactions that may impact sulfur speciation and assimilation catalyzed by sulfurtransferases (STR) (
      • Walsh B.J.C.
      • Brito J.A.
      • Giedroc D.P.
      Hydrogen sulfide signaling and enzymology.
      ), peroxiredoxins (Prx) (
      • Cuevasanta E.
      • Reyes A.M.
      • Zeida A.
      • Mastrogiovanni M.
      • De Armas M.I.
      • Radi R.
      • Alvarez B.
      • Trujillo M.
      Kinetics of formation and reactivity of the persulfide in the one-cysteine peroxiredoxin from Mycobacterium tuberculosis.
      ), or other thiol-containing enzymes or via nonenzymatic interconversion among organic LMW thiol/persulfides or inorganic RSS. D, enzymatic clearance of RSS known to occur in bacteria (
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ,
      • Libiad M.
      • Yadav P.K.
      • Vitvitsky V.
      • Martinov M.
      • Banerjee R.
      Organization of the human mitochondrial hydrogen sulfide oxidation pathway.
      ,
      • Motl N.
      • Skiba M.A.
      • Kabil O.
      • Smith J.L.
      • Banerjee R.
      Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase-rhodanese fusion protein functions in sulfur assimilation.
      ,
      • Shen J.
      • Keithly M.E.
      • Armstrong R.N.
      • Higgins K.A.
      • Edmonds K.A.
      • Giedroc D.P.
      Staphylococcus aureus CstB is a novel multidomain persulfide dioxygenase-sulfurtransferase involved in hydrogen sulfide detoxification.
      ). Red sulfurs indicate inorganic sulfur additions. Characterized enzymes are represented by blue ovals, whereas incompletely understood enzymatic reactions are shown as gray ovals and dashed arrows. CoAPR, CoA persulfide reductase; CstB, S. aureus cst operon-encoded persulfide dioxygenase-rhodanese fusion; 3MST, 3-mercaptopyruvate sulfurtransferase; PDO, persulfide dioxygenase; PRF, persulfide dioxygenase-rhodanese fusion; Q/QH2, quinone; SQR, sulfide:quinone oxidoreductase.
      An important enzymatic route to the biogenesis of RSS in bacteria is the sulfide:quinone oxidoreductase (SQR) (
      • Libiad M.
      • Yadav P.K.
      • Vitvitsky V.
      • Martinov M.
      • Banerjee R.
      Organization of the human mitochondrial hydrogen sulfide oxidation pathway.
      ,
      • Shen J.
      • Peng H.
      • Zhang Y.
      • Trinidad J.C.
      • Giedroc D.P.
      Staphylococcus aureus sqr encodes a type II sulfide:quinone oxidoreductase and impacts reactive sulfur speciation in cells.
      ,
      • Duzs Á.
      • Tóth A.
      • Németh B.
      • Balogh T.
      • Kós P.B.
      • Rákhely G.
      A novel enzyme of type VI sulfide:quinone oxidoreductases in purple sulfur photosynthetic bacteria.
      ,
      • Landry A.P.
      • Moon S.
      • Kim H.
      • Yadav P.K.
      • Guha A.
      • Cho U.-S.
      • Banerjee R.
      A catalytic trisulfide in human sulfide quinone oxidoreductase catalyzes coenzyme a persulfide synthesis and inhibits butyrate oxidation.
      ). SQR catalyzes the two-electron oxidation of H2S to sulfane sulfur fixed as organic and inorganic per- and polysulfides (Fig. 4B), concomitant with reduction of the quinone pool (
      • Walsh B.J.C.
      • Brito J.A.
      • Giedroc D.P.
      Hydrogen sulfide signaling and enzymology.
      ). This enzyme may well provide a source of electrons for the alternative cytochrome bd oxidase in organisms that encode this alternate oxidase, analogous to that observed for SQR with complex III/IV when the concentration of H2S is low (Fig. 3B) (
      • Libiad M.
      • Yadav P.K.
      • Vitvitsky V.
      • Martinov M.
      • Banerjee R.
      Organization of the human mitochondrial hydrogen sulfide oxidation pathway.
      ). In organisms (e.g. Enteroccocus faecalis) that do not appear to encode an SQR but where RSS have been detected and quantified (
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ), the mechanism of RSS biogenesis is not known, and may well suggest a role for nonenzymatic or as yet uncharacterized enzymatic processes in these organisms. In addition to SQR, recent work in A. baumannii reveals that 3MST may also contribute to pools of LMW persulfides, although there are clearly other contributors (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ).

      Sulfurtransferases

      Major structural classes of sulfurtransferases (STRs) adopt either a rhodanese or TusA (tRNA 2-thiouridine–synthesizing protein A)-like fold (
      • Walsh B.J.C.
      • Brito J.A.
      • Giedroc D.P.
      Hydrogen sulfide signaling and enzymology.
      ) and harbor an active-site cysteine that is known or projected to function in interdomain or intermolecular persulfide transfer, termed transsulfuration (Fig. 4C) (
      • Mueller E.G.
      Trafficking in persulfides: delivering sulfur in biosynthetic pathways.
      ). Although rhodanese domains were originally believed to function in cyanide (CN) detoxification by forming thiocyanide (SCN) (
      • Cipollone R.
      • Ascenzi P.
      • Visca P.
      Common themes and variations in the rhodanese superfamily.
      ), it is well-established that Fe-S cluster biogenesis, molybdenum cofactor biosynthesis, 2-thiouridine synthesis, and thiamine pyrophosphate biosynthesis are known or proposed to use STRs as persulfide transfer catalysts (
      • Dahl J.U.
      • Radon C.
      • Bühning M.
      • Nimtz M.
      • Leichert L.I.
      • Denis Y.
      • Jourlin-Castelli C.
      • Iobbi-Nivol C.
      • Méjean V.
      • Leimkühler S.
      The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis.
      ,
      • Dahl J.U.
      • Urban A.
      • Bolte A.
      • Sriyabhaya P.
      • Donahue J.L.
      • Nimtz M.
      • Larson T.J.
      • Leimkühler S.
      The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli.
      ,
      • Palmer L.D.
      • Leung M.H.
      • Downs D.M.
      The cysteine desulfhydrase CdsH is conditionally required for sulfur mobilization to the thiamine thiazole in Salmonella enterica.
      ,
      • Shi R.
      • Proteau A.
      • Villarroya M.
      • Moukadiri I.
      • Zhang L.
      • Trempe J.F.
      • Matte A.
      • Armengod M.E.
      • Cygler M.
      Structural basis for Fe-S cluster assembly and tRNA thiolation mediated by IscS protein-protein interactions.
      ,
      • Shigi N.
      Recent advances in our understanding of the biosynthesis of sulfur modifications in tRNAs.
      ). Such “targeted” transsulfuration reactions require specific, albeit likely transient, interactions between donor and acceptor and an exposed active site, as described for TSTD1 and thioredoxin in colon epithelial cells (
      • Libiad M.
      • Motl N.
      • Akey D.L.
      • Sakamoto N.
      • Fearon E.R.
      • Smith J.L.
      • Banerjee R.
      Thiosulfate sulfurtransferase-like domain-containing 1 protein interacts with thioredoxin.
      ).
      “Orphan” STRs, which we define as not yet connected to any biosynthetic pathway, particularly those regulated by RSS sensors in bacteria, may well play roles in sulfide detoxification or assimilation (
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ,
      • Higgins K.A.
      • Peng H.
      • Luebke J.L.
      • Chang F.M.
      • Giedroc D.P.
      Conformational analysis and chemical reactivity of the multidomain sulfurtransferase, Staphylococcus aureus CstA.
      ), but their biological functions remain enigmatic. This remains a significant challenge in the field. RSS sensor–regulated STRs are often kinetically characterized in vitro as sulfurtransferases from a thiosulfate donor to a CN acceptor; however, their physiological donors and acceptors, whether they be small molecules or proteins, have generally not been identified, and any role in targeted transsulfuration has not been established (Fig. 4C). Recently, a single cysteine peroxiredoxin (a major H2O2-detoxifying enzyme) characterized by a long-lived sulfenylated intermediate was shown to rapidly react with H2S to form a protein persulfide, which participated in persulfide transfer to a thiol acceptor (
      • Cuevasanta E.
      • Reyes A.M.
      • Zeida A.
      • Mastrogiovanni M.
      • De Armas M.I.
      • Radi R.
      • Alvarez B.
      • Trujillo M.
      Kinetics of formation and reactivity of the persulfide in the one-cysteine peroxiredoxin from Mycobacterium tuberculosis.
      ). This suggests that peroxiredoxins may function in transsulfuration, but this requires further investigation (Fig. 4C). Similarly, the extent to which small-molecule RSS species, particularly those containing sulfane sulfur, participate in transsulfuration reactions with each other, LMW thiols, or even protein thiols is largely unknown (Fig. 4C).

      RSS clearance enzymes

      In addition to the biogenesis of RSS, a number of bacterial enzymes have been characterized that function in their clearance (Fig. 4D). A well-known player in the clearance of organic persulfides is persulfide dioxygenase (PDO), which harbors a mononuclear, nonheme FeII center (
      • Libiad M.
      • Yadav P.K.
      • Vitvitsky V.
      • Martinov M.
      • Banerjee R.
      Organization of the human mitochondrial hydrogen sulfide oxidation pathway.
      ,
      • McCoy J.G.
      • Bingman C.A.
      • Bitto E.
      • Holdorf M.M.
      • Makaroff C.A.
      • Phillips Jr., G.N.
      Structure of an ETHE1-like protein from Arabidopsis thaliana.
      ,
      • Motl N.
      • Skiba M.A.
      • Kabil O.
      • Smith J.L.
      • Banerjee R.
      Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase-rhodanese fusion protein functions in sulfur assimilation.
      ,
      • Shen J.
      • Keithly M.E.
      • Armstrong R.N.
      • Higgins K.A.
      • Edmonds K.A.
      • Giedroc D.P.
      Staphylococcus aureus CstB is a novel multidomain persulfide dioxygenase-sulfurtransferase involved in hydrogen sulfide detoxification.
      ,
      • Sattler S.A.
      • Wang X.
      • Lewis K.M.
      • DeHan P.J.
      • Park C.M.
      • Xin Y.
      • Liu H.
      • Xian M.
      • Xun L.
      • Kang C.
      Characterizations of two bacterial persulfide dioxygenases of the metallo-β-lactamase superfamily.
      ). In bacteria, PDOs have been characterized as single or multidomain enzymes, and the presence of additional domains appears to impact the distribution of products. All PDOs, regardless of their domain organization, use molecular oxygen to oxidize the terminal sulfur of an RSSH substrate to sulfite, which, for a single-domain PDO, is the final product. Some PDOs have an appended STR domain, and these have been designated PDO-rhodanese fusion proteins (PRFs) (Fig. 4D). In the case of the PRF characterized in Burkholderia phytofirmans, the C-terminal rhodanese domain generates the GSH persulfide substrate that the PDO domain then oxidizes to sulfite (
      • Motl N.
      • Skiba M.A.
      • Kabil O.
      • Smith J.L.
      • Banerjee R.
      Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase-rhodanese fusion protein functions in sulfur assimilation.
      ). In contrast, the multidomain PRF CstB from S. aureus oxidizes two equivalents of persulfide substrate to thiosulfate as the final product; the C-terminal rhodanese domain also possesses transsulfuration and thiosulfate transferase activity (
      • Shen J.
      • Keithly M.E.
      • Armstrong R.N.
      • Higgins K.A.
      • Edmonds K.A.
      • Giedroc D.P.
      Staphylococcus aureus CstB is a novel multidomain persulfide dioxygenase-sulfurtransferase involved in hydrogen sulfide detoxification.
      ). In contrast to the oxidative chemistry of PDOs, E. faecalis encodes a CoA disulfide reductase-rhodanese homology domain fusion protein (CoADR-RHD) that specifically reduces CoA persulfide to form the reduced thiol and H2S and is thus a CoA persulfide reductase (CoAPR) (Fig. 4D) (
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ,
      • Lee K.H.
      • Humbarger S.
      • Bahnvadia R.
      • Sazinsky M.H.
      • Crane 3rd, E.J.
      Characterization of the mechanism of the NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4: formation of a productive NADH-enzyme complex and its role in the general mechanism of NADH and FAD-dependent enzymes.
      ,
      • Warner M.D.
      • Lukose V.
      • Lee K.H.
      • Lopez K.
      • M H.S.
      • Crane 3rd, E.J.
      Characterization of an NADH-dependent persulfide reductase from Shewanella loihica PV-4: implications for the mechanism of sulfur respiration via FAD-dependent enzymes.
      ,
      • Wallen J.R.
      • Mallett T.C.
      • Boles W.
      • Parsonage D.
      • Furdui C.M.
      • Karplus P.A.
      • Claiborne A.
      Crystal structure and catalytic properties of Bacillus anthracis CoADR-RHD: implications for flavin-linked sulfur trafficking.
      ).

      Regulatory sensing of RSS in bacteria

      The discovery of endogenous H2S production and pathways for the biogenesis and clearance of RSS in bacteria requires a mechanism to establish cellular H2S/RSS homeostasis. This is mediated by RSS sensors (Fig. 1A, bottom). We and others have discovered and characterized structurally diverse transcriptional regulators that react with RSS to drive transcriptional derepression or activation of genes encoding common sulfide detoxification or oxidation enzymes described above. These RSS sensors are widespread and have been identified in both Gram-positive and Gram-negative organisms. They include CstR from S. aureus (
      • Grossoehme N.
      • Kehl-Fie T.E.
      • Ma Z.
      • Adams K.W.
      • Cowart D.M.
      • Scott R.A.
      • Skaar E.P.
      • Giedroc D.P.
      Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.
      ,
      • Luebke J.L.
      • Shen J.
      • Bruce K.E.
      • Kehl-Fie T.E.
      • Peng H.
      • Skaar E.P.
      • Giedroc D.P.
      The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.
      ) and E. faecalis (
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ), SqrR from Rhodobacter capsulatus (
      • Shimizu T.
      • Shen J.
      • Fang M.
      • Zhang Y.
      • Hori K.
      • Trinidad J.C.
      • Bauer C.E.
      • Giedroc D.P.
      • Masuda S.
      Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis.
      ,
      • Capdevila D.A.
      • Walsh B.J.C.
      • Zhang Y.
      • Dietrich C.
      • Gonzalez-Gutierrez G.
      • Giedroc D.P.
      Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator.
      ), the SqrR homolog BigR from Xylella fastidiosa (
      • Barbosa R.L.
      • Benedetti C.E.
      BigR, a transcriptional repressor from plant-associated bacteria, regulates an operon implicated in biofilm growth.
      ,
      • Guimarães B.G.
      • Barbosa R.L.
      • Soprano A.S.
      • Campos B.M.
      • de Souza T.A.
      • Tonoli C.C.C.
      • Leme A.F.P.
      • Murakami M.T.
      • Benedetti C.E.
      Plant pathogenic bacteria utilize biofilm growth-associated repressor (BigR), a novel winged-helix redox switch, to control hydrogen sulfide detoxification under hypoxia.
      ,
      • de Lira N.P.V.
      • Pauletti B.A.
      • Marques A.C.
      • Perez C.A.
      • Caserta R.
      • de Souza A.A.
      • Vercesi A.E.
      • Paes Leme A.F.
      • Benedetti C.E.
      BigR is a sulfide sensor that regulates a sulfur transferase/dioxygenase required for aerobic respiration of plant bacteria under sulfide stress.
      ) and A. baumannii (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ), and FisR from Cupriavidus pinatubonensis (
      • Li H.
      • Li J.
      • Lu C.
      • Xia Y.
      • Xin Y.
      • Liu H.
      • Xun L.
      • Liu H.
      FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.
      ) and A. baumannii (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ).

      CstR

      The CsoR-like sulfurtransferase repressor, CstR, is a member of the CsoR (copper-sensitive operon repressor) family of transcriptional repressors (
      • Liu T.
      • Ramesh A.
      • Ma Z.
      • Ward S.K.
      • Zhang L.
      • George G.N.
      • Talaat A.M.
      • Sacchettini J.C.
      • Giedroc D.P.
      CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator.
      ) and was first discovered in S. aureus (
      • Grossoehme N.
      • Kehl-Fie T.E.
      • Ma Z.
      • Adams K.W.
      • Cowart D.M.
      • Scott R.A.
      • Skaar E.P.
      • Giedroc D.P.
      Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.
      ,
      • Luebke J.L.
      • Shen J.
      • Bruce K.E.
      • Kehl-Fie T.E.
      • Peng H.
      • Skaar E.P.
      • Giedroc D.P.
      The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.
      ). S. aureus CstR regulates the cst operon encoding a multidomain STR (CstA), a PRF (CstB), and a type II SQR, rather analogous to the well-studied mitochondrial sulfide detoxification system (
      • Libiad M.
      • Yadav P.K.
      • Vitvitsky V.
      • Martinov M.
      • Banerjee R.
      Organization of the human mitochondrial hydrogen sulfide oxidation pathway.
      ,
      • Shen J.
      • Peng H.
      • Zhang Y.
      • Trinidad J.C.
      • Giedroc D.P.
      Staphylococcus aureus sqr encodes a type II sulfide:quinone oxidoreductase and impacts reactive sulfur speciation in cells.
      ,
      • Higgins K.A.
      • Peng H.
      • Luebke J.L.
      • Chang F.M.
      • Giedroc D.P.
      Conformational analysis and chemical reactivity of the multidomain sulfurtransferase, Staphylococcus aureus CstA.
      ,
      • Shen J.
      • Keithly M.E.
      • Armstrong R.N.
      • Higgins K.A.
      • Edmonds K.A.
      • Giedroc D.P.
      Staphylococcus aureus CstB is a novel multidomain persulfide dioxygenase-sulfurtransferase involved in hydrogen sulfide detoxification.
      ). The enzymes encoded by the S. aureus cst operon oxidize sulfide to thiosulfate via persulfide intermediates and have been extensively reviewed elsewhere (
      • Walsh B.J.C.
      • Brito J.A.
      • Giedroc D.P.
      Hydrogen sulfide signaling and enzymology.
      ). Recently, we characterized CstR from a second human pathogen E. faecalis that regulates a cst-like operon encoding two orphan STRs and a CoAPR (Fig. 5A, left) (
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ). CstR is homotetrameric in solution and is anticipated to harbor four peripherally arranged dithiol “sensing” sites found between protomers in a dimer-of-dimers D2-symmetric architecture, like CsoR (Fig. 5A, middle) (
      • Chang F.M.
      • Coyne H.J.
      • Cubillas C.
      • Vinuesa P.
      • Fang X.
      • Ma Z.
      • Ma D.
      • Helmann J.D.
      • García-de los Santos A.
      • Wang Y.X.
      • Dann 3rd, C.E.
      • Giedroc D.P.
      Cu(I)-mediated allosteric switching in a copper-sensing operon repressor (CsoR).
      ). CstR represses transcription in the reduced state, whereas reaction with sulfane sulfur–containing RSS, but not H2S itself, results in a mixture of di-, tri-, and tetrasulfide interprotomer cross-links that negatively regulates DNA operator-promoter binding, allowing for transcription initiation (Fig. 5A, right) (
      • Luebke J.L.
      • Shen J.
      • Bruce K.E.
      • Kehl-Fie T.E.
      • Peng H.
      • Skaar E.P.
      • Giedroc D.P.
      The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.
      ). In both S. aureus and E. faecalis, the operons are also inducible by the nitroxyl (HNO) donor Angeli's salt, but not by an NO· donor (
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ,
      • Peng H.
      • Shen J.
      • Edmonds K.A.
      • Luebke J.L.
      • Hickey A.K.
      • Palmer L.D.
      • Chang F.J.
      • Bruce K.A.
      • Kehl-Fie T.E.
      • Skaar E.P.
      • Giedroc D.P.
      Sulfide homeostasis and nitroxyl intersect via formation of reactive sulfur species in Staphylococcus aureus.
      ). In S. aureus this may be the result of increased cellular per- and polysulfides after the addition of nitroxyl to cells, supporting the notion of H2S/NO· cross-talk in this organism (Fig. 2B). Although CstR appears selective for H2S and RSS in cell-based transcription reactions (
      • Luebke J.L.
      • Shen J.
      • Bruce K.E.
      • Kehl-Fie T.E.
      • Peng H.
      • Skaar E.P.
      • Giedroc D.P.
      The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.
      ), the structure/reactivity factors that enforce this apparent specificity are not yet known.
      Figure thumbnail gr5
      Figure 5Regulated operons, modes of regulation, structure, and RSS reactivity products of bacterial RSS sensors. A, CstR in its reduced form transcriptionally represses the cst and cst-like operons in S. aureus and E. faecalis, respectively (left) (
      • Luebke J.L.
      • Shen J.
      • Bruce K.E.
      • Kehl-Fie T.E.
      • Peng H.
      • Skaar E.P.
      • Giedroc D.P.
      The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.
      ). A structural model of CstR using Protein Data Bank entry 5FMN as a template reveals an all α-helical protein with four peripheral dithiol-sensing sites in the tetrameric structure (middle). The reaction products of CstR with RSS reveal a mixture of di-, tri-, and tetrasulfide interprotomer linkages (right). B, SqrR (operon not shown), like its homologs BigR and PigS, function as transcriptional repressors in their reduced forms and regulate the expression of typical sulfide oxidation and detoxification enzymes (left) (
      • Shimizu T.
      • Shen J.
      • Fang M.
      • Zhang Y.
      • Hori K.
      • Trinidad J.C.
      • Bauer C.E.
      • Giedroc D.P.
      • Masuda S.
      Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis.
      ,
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ,
      • de Lira N.P.V.
      • Pauletti B.A.
      • Marques A.C.
      • Perez C.A.
      • Caserta R.
      • de Souza A.A.
      • Vercesi A.E.
      • Paes Leme A.F.
      • Benedetti C.E.
      BigR is a sulfide sensor that regulates a sulfur transferase/dioxygenase required for aerobic respiration of plant bacteria under sulfide stress.
      ,
      • Gristwood T.
      • McNeil M.B.
      • Clulow J.S.
      • Salmond G.P.
      • Fineran P.C.
      PigS and PigP regulate prodigiosin biosynthesis in Serratia via differential control of divergent operons, which include predicted transporters of sulfur-containing molecules.
      ). The homodimeric structure of SqrR reveals a pair of dithiol-sensing sites (middle, Protein Data Bank entry 6O8L), which readily form intraprotomer sulfur bridges of four (SqrR) or five (BigR) sulfur atoms upon reaction with RSS (right) (
      • Capdevila D.A.
      • Walsh B.J.C.
      • Zhang Y.
      • Dietrich C.
      • Gonzalez-Gutierrez G.
      • Giedroc D.P.
      Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator.
      ). C, FisR is a σ54-dependent transcriptional activator and activates the expression of a sulfide detoxification operon that is similar to that encoded by the cst operon (left). FisR is organized into three domains (regulatory, ATPase, and DNA-binding domain), but to date, there are no structures of a functionally characterized RSS-sensing FisR (middle). C. pinatubonensis FisR reacts with RSS in vitro to form a mixture of di- and tetrasulfide linkages that weakly stimulate the ATPase activity, resulting in transcriptional activation from σ54-RNA polymerase (RNAP)-transcribed promoters (right) (
      • Li H.
      • Li J.
      • Lu C.
      • Xia Y.
      • Xin Y.
      • Liu H.
      • Xun L.
      • Liu H.
      FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.
      ). The regulatory mechanism operative in A. baumannii FisR has not yet been determined (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ).

      SqrR and homologs

      The sulfide:quinone reductase repressor, SqrR, was originally characterized in the photosynthetic bacterium R. capsulatus and is responsible for the regulation of 45% of all sulfide-responsive genes in this organism, including an SQR (
      • Shimizu T.
      • Shen J.
      • Fang M.
      • Zhang Y.
      • Hori K.
      • Trinidad J.C.
      • Bauer C.E.
      • Giedroc D.P.
      • Masuda S.
      Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis.
      ). SqrR is a member of the arsenic repressor (ArsR) superfamily (
      • Busenlehner L.S.
      • Pennella M.A.
      • Giedroc D.P.
      The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance.
      ) in striking structural contrast to CstR. SqrR adopts the ArsR family α1-α2-α3-α4-β1-β2-α5 “winged-helical” dimeric fold, where one Cys in the α2 helix and one Cys in the α5 helix from the same subunit create a pair of dithiol RSS-sensing sites on the dimer (Fig. 5B, middle) (
      • Capdevila D.A.
      • Walsh B.J.C.
      • Zhang Y.
      • Dietrich C.
      • Gonzalez-Gutierrez G.
      • Giedroc D.P.
      Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator.
      ). Other RSS-responsive ArsR family repressors include the biofilm growth-associated repressor BigR, characterized in X. fastidiosa (
      • Barbosa R.L.
      • Benedetti C.E.
      BigR, a transcriptional repressor from plant-associated bacteria, regulates an operon implicated in biofilm growth.
      ,
      • Guimarães B.G.
      • Barbosa R.L.
      • Soprano A.S.
      • Campos B.M.
      • de Souza T.A.
      • Tonoli C.C.C.
      • Leme A.F.P.
      • Murakami M.T.
      • Benedetti C.E.
      Plant pathogenic bacteria utilize biofilm growth-associated repressor (BigR), a novel winged-helix redox switch, to control hydrogen sulfide detoxification under hypoxia.
      ,
      • de Lira N.P.V.
      • Pauletti B.A.
      • Marques A.C.
      • Perez C.A.
      • Caserta R.
      • de Souza A.A.
      • Vercesi A.E.
      • Paes Leme A.F.
      • Benedetti C.E.
      BigR is a sulfide sensor that regulates a sulfur transferase/dioxygenase required for aerobic respiration of plant bacteria under sulfide stress.
      ) and A. baumannii (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ), and PigS characterized in Serratia spp. (
      • Gristwood T.
      • McNeil M.B.
      • Clulow J.S.
      • Salmond G.P.
      • Fineran P.C.
      PigS and PigP regulate prodigiosin biosynthesis in Serratia via differential control of divergent operons, which include predicted transporters of sulfur-containing molecules.
      ). A. baumannii BigR regulates a secondary RSS detoxification system that includes a second PDO and two transmembrane proteins proposed to be involved in the transport of sulfur-containing molecules (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ,
      • Gristwood T.
      • McNeil M.B.
      • Clulow J.S.
      • Salmond G.P.
      • Fineran P.C.
      PigS and PigP regulate prodigiosin biosynthesis in Serratia via differential control of divergent operons, which include predicted transporters of sulfur-containing molecules.
      ). Although PigS has not been functionally characterized as an RSS sensor, it regulates several enzymes known to function in H2S/RSS clearance, including a single-domain PDO and a CoAPR encoded by coaP (Fig. 5B, left) (
      • Gristwood T.
      • McNeil M.B.
      • Clulow J.S.
      • Salmond G.P.
      • Fineran P.C.
      PigS and PigP regulate prodigiosin biosynthesis in Serratia via differential control of divergent operons, which include predicted transporters of sulfur-containing molecules.
      ). It is interesting to note that PigS and its regulon are part of the larger PigP regulon involved in the biosynthesis of the antibiotic prodigiosin (Pig), thus implying a regulatory connection between antibiotic biosynthesis and H2S/RSS homeostasis. The ArsR-family RSS sensors that have been functionally characterized behave analogously to CstR, functioning as repressors in the reduced state and dissociating from the DNA upon reaction with sulfane sulfur–containing RSS, to readily form nearly exclusively tetrasulfide (SqrR) and pentasulfide (BigR) bridges, respectively (Fig. 5B, right) (
      • Capdevila D.A.
      • Walsh B.J.C.
      • Zhang Y.
      • Dietrich C.
      • Gonzalez-Gutierrez G.
      • Giedroc D.P.
      Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator.
      ). In contrast to CstR, these (poly)sulfur bridges are formed within a subunit, and although other linkages are made, they are far less abundant compared with the mixture of products found in CstR (
      • Luebke J.L.
      • Shen J.
      • Bruce K.E.
      • Kehl-Fie T.E.
      • Peng H.
      • Skaar E.P.
      • Giedroc D.P.
      The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.
      ).
      Recent work from our laboratory utilized SqrR as a model dithiol transcriptional regulator to investigate the structural and reactivity features that govern its oxidant selectivity and specificity (
      • Capdevila D.A.
      • Walsh B.J.C.
      • Zhang Y.
      • Dietrich C.
      • Gonzalez-Gutierrez G.
      • Giedroc D.P.
      Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator.
      ). Indeed, SqrR is specific for sulfane sulfur and only forms a disulfide when treated with potent diazirene electrophile TMAD (diamide), but not with more common cellular oxidants including GSH disulfide or H2O2. Whereas this low reactivity toward cellular oxidants can be partially explained by the relatively high apparent pKa of the dithiol pair, the high selectivity toward RSS is enforced by structural features of SqrR in various oxidation states. These structures reveal a high energetic barrier to form the disulfide because of large rearrangements that must occur in order to form the disulfide; in addition, the disulfide is not on pathway to form the major tetrasulfide product. In contrast, formation of the tetrasulfide does not require large structural rearrangements; on the contrary, this linkage results in the collapse of the dithiol pocket that completely shields the tetrasulfide linkage from solvent. This study demonstrates that SqrR-like dithiol-based repressors achieve high RSS specificity from the conformational landscape of the protein ensemble, which favors installation of a PTM that minimizes local structural frustration (
      • Capdevila D.A.
      • Walsh B.J.C.
      • Zhang Y.
      • Dietrich C.
      • Gonzalez-Gutierrez G.
      • Giedroc D.P.
      Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator.
      ). It will be interesting to determine whether these principles apply to other structural classes of RSS sensors or if there are additional determinants that dictate their specificity.

      FisR

      A third structural class of RSS-sensing transcriptional regulators first characterized in C. pinatubonensis (
      • Li H.
      • Li J.
      • Lu C.
      • Xia Y.
      • Xin Y.
      • Liu H.
      • Xun L.
      • Liu H.
      FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.
      ), and more recently in A. baumannii (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ), is FisR (Fis family transcriptional regulator). In both bacteria, FisR transcriptionally activates the expression of a PDO and SQR and a putative sulfite/sulfonate effluxer, TauE (
      • Weinitschke S.
      • Denger K.
      • Cook A.M.
      • Smits T.H.M.
      The DUF81 protein TauE in Cupriavidus necator H16, a sulfite exporter in the metabolism of C2 sulfonates.
      ), in only A. baumannii (Fig. 5C, left). In contrast to CstR and SqrR-like RSS sensors, FisR is a canonical σ54-dependent transcriptional activator that harbors an N-terminal regulatory domain, a central AAA+ ATPase domain, and a C-terminal DNA-binding domain (Fig. 5C, middle) (
      • Studholme D.J.
      • Dixon R.
      Domain architectures of σ54-dependent transcriptional activators.
      ). In C. pinatubonensis FisR, reaction with inorganic RSS appears to result in the formation of di- and tetrasulfide cross-links between two cysteine residues in the regulatory domain, which in turn stimulates the ATPase activity of the central AAA+ domain, which likely activates hexameric assembly and promoter melting by σ54-RNA polymerase holoenzyme (Fig. 5C, right) (
      • Li H.
      • Li J.
      • Lu C.
      • Xia Y.
      • Xin Y.
      • Liu H.
      • Xun L.
      • Liu H.
      FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.
      ). In A. baumannii FisR, these cysteines are not present, and as a result, H2S/RSS is likely sensed using an alternate mechanism (
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ), which includes heme-based (
      • Vitvitsky V.
      • Yadav P.K.
      • Kurthen A.
      • Banerjee R.
      Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides.
      ,
      • Bostelaar T.
      • Vitvitsky V.
      • Kumutima J.
      • Lewis B.E.
      • Yadav P.K.
      • Brunold T.C.
      • Filipovic M.
      • Lehnert N.
      • Stemmler T.L.
      • Banerjee R.
      Hydrogen sulfide oxidation by myoglobin.
      ,
      • Ruetz M.
      • Kumutima J.
      • Lewis B.E.
      • Filipovic M.R.
      • Lehnert N.
      • Stemmler T.L.
      • Banerjee R.
      A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin.
      ,
      • Vitvitsky V.
      • Miljkovic J.L.
      • Bostelaar T.
      • Adhikari B.
      • Yadav P.K.
      • Steiger A.K.
      • Torregrossa R.
      • Pluth M.D.
      • Whiteman M.
      • Banerjee R.
      • Filipovic M.R.
      Cytochrome c reduction by H2S potentiates sulfide signaling.
      ,
      • Nelp M.T.
      • Zheng V.
      • Davis K.M.
      • Stiefel K.J.E.
      • Groves J.T.
      Potent activation of indoleamine 2,3-dioxygenase by polysulfides.
      ) and mononuclear, nonheme Fe-based RSS-sensing regulatory models (
      • D'Autreaux B.
      • Tucker N.P.
      • Dixon R.
      • Spiro S.
      A non-haem iron centre in the transcription factor NorR senses nitric oxide.
      ,
      • Tucker N.P.
      • D'Autreaux B.
      • Yousafzai F.K.
      • Fairhurst S.A.
      • Spiro S.
      • Dixon R.
      Analysis of the nitric oxide-sensing non-heme iron center in the NorR regulatory protein.
      ,
      • Yang B.
      • Nie X.
      • Xiao Y.
      • Gu Y.
      • Jiang W.
      • Yang C.
      Ferrous-iron-activated transcriptional factor AdhR regulates redox homeostasis in Clostridium beijerinckii.
      ).

      Chemical tools for generation, detection, and quantification of RSS

      To understand the role of H2S and RSS in signaling at the host-pathogen interface, tools must be available that allow for the generation, detection, and quantification of these species in vivo. The type and number of molecular probes used for the generation of H2S and RSS have substantially increased over the past several years, and they are now being used in bacteria to provide critical insights into H2S signaling in these organisms (
      • Toliver-Kinsky T.
      • Cui W.
      • Törö G.
      • Lee S.-J.
      • Shatalin K.
      • Nudler E.
      • Szabo C.
      H2S, a bacterial defense mechanism against the host immune response.
      ,
      • Saini V.
      • Chinta K.C.
      • Reddy V.P.
      • Glasgow J.N.
      • Stein A.
      • Lamprecht D.A.
      • Rahman M.A.
      • Mackenzie J.S.
      • Truebody B.E.
      • Adamson J.H.
      • Kunota T.T.R.
      • Bailey S.M.
      • Moellering D.R.
      • Lancaster Jr., J.R.
      • Steyn A.J.C.
      Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration, growth and pathogenesis.
      ,
      • Shukla P.
      • Khodade V.S.
      • SharathChandra M.
      • Chauhan P.
      • Mishra S.
      • Siddaramappa S.
      • Pradeep B.E.
      • Singh A.
      • Chakrapani H.
      “On demand” redox buffering by H2S contributes to antibiotic resistance revealed by a bacteria-specific H2S donor.
      ). Fluorescence-based probes provide rapid and sensitive detection of H2S or sulfane sulfur with several options now commercially available. In addition, recent efforts to quantify H2S and RSS in complex cellular mixtures have provided new insights into this process. As many of these molecular tools have been extensively reviewed elsewhere (
      • Bora P.
      • Chauhan P.
      • Pardeshi K.A.
      • Chakrapani H.
      Small molecule generators of biologically reactive sulfur species.
      ,
      • Levinn C.M.
      • Cerda M.M.
      • Pluth M.D.
      Activatable small-molecule hydrogen sulfide donors.
      ,
      • Lin V.S.
      • Chen W.
      • Xian M.
      • Chang C.J.
      Chemical probes for molecular imaging and detection of hydrogen sulfide and reactive sulfur species in biological systems.
      ,
      • Powell C.R.
      • Dillon K.M.
      • Matson J.B.
      A review of hydrogen sulfide (H2S) donors: chemistry and potential therapeutic applications.
      ,
      • Takano Y.
      • Echizen H.
      • Hanaoka K.
      Fluorescent probes and selective inhibitors for biological studies of hydrogen sulfide- and polysulfide-mediated signaling.
      ,
      • Xu S.
      • Hamsath A.
      • Neill D.L.
      • Wang Y.
      • Yang C.T.
      • Xian M.
      Strategies for the design of donors and precursors of reactive sulfur species.
      ,
      • Jiao X.
      • Li Y.
      • Niu J.
      • Xie X.
      • Wang X.
      • Tang B.
      Small-molecule fluorescent probes for imaging and detection of reactive oxygen, nitrogen, and sulfur species in biological systems.
      ), here we provide only a summary of the available approaches, while pointing out specific challenges to their use.

      H2S and RSS donors

      H2S donors largely fall into three main classes. These are hydrolysis-based, thiol-dependent, and carbonyl sulfide (COS)-based donors (Fig. 6A). Hydrolysis-based donors function over a wide range of pH, with GYY4137 as a widely used and commercially available donor employed by several groups to study the mechanism of H2S cytoprotection in bacteria (
      • Toliver-Kinsky T.
      • Cui W.
      • Törö G.
      • Lee S.-J.
      • Shatalin K.
      • Nudler E.
      • Szabo C.
      H2S, a bacterial defense mechanism against the host immune response.
      ,
      • Saini V.
      • Chinta K.C.
      • Reddy V.P.
      • Glasgow J.N.
      • Stein A.
      • Lamprecht D.A.
      • Rahman M.A.
      • Mackenzie J.S.
      • Truebody B.E.
      • Adamson J.H.
      • Kunota T.T.R.
      • Bailey S.M.
      • Moellering D.R.
      • Lancaster Jr., J.R.
      • Steyn A.J.C.
      Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration, growth and pathogenesis.
      ,
      • Li L.
      • Whiteman M.
      • Guan Y.Y.
      • Neo K.L.
      • Cheng Y.
      • Lee S.W.
      • Zhao Y.
      • Baskar R.
      • Tan C.H.
      • Moore P.K.
      Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide.
      ). Concerns over the relatively slow release rates have led to second-generation GYY4137 derivatives, including JK-2, that more efficiently release H2S at physiological pH (
      • Kang J.
      • Li Z.
      • Organ C.L.
      • Park C.-M.
      • Yang C-T.
      • Pacheco A.
      • Wang D.
      • Lefer D.J.
      • Xian M.
      pH-controlled hydrogen sulfide release for myocardial ischemia-reperfusion injury.
      ). The use of hydrolysis-based donors requires careful consideration of the pH dependence and kinetics of H2S release, and many donors are not commercially available. Thiol-dependent H2S donors are attractive tools because of their use of typically cell-abundant cellular reducing thiols, including GSH and cysteine (Fig. 6A). However, some of these probes are quite slow, require high concentrations of thiol, or are activated by a specific thiol (
      • Levinn C.M.
      • Cerda M.M.
      • Pluth M.D.
      Activatable small-molecule hydrogen sulfide donors.
      ). As the type and concentrations of thiols in bacteria have only recently been investigated and only in a small sampling of bacteria (
      • Shen J.
      • Walsh B.J.C.
      • Flores-Mireles A.L.
      • Peng H.
      • Zhang Y.
      • Zhang Y.
      • Trinidad J.C.
      • Hultgren S.J.
      • Giedroc D.P.
      Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis.
      ,
      • Walsh B.J.C.
      • Wang J.
      • Edmonds K.A.
      • Palmer L.D.
      • Zhang Y.
      • Trinidad J.C.
      • Skaar E.P.
      • Giedroc D.P.
      The response of Acinetobacter baumannii to hydrogen sulfide reveals two independent persulfide sensing systems and a connection to biofilm regulation.
      ,
      • Peng H.
      • Shen J.
      • Edmonds K.A.
      • Luebke J.L.
      • Hickey A.K.
      • Palmer L.D.
      • Chang F.J.
      • Bruce K.A.
      • Kehl-Fie T.E.
      • Skaar E.P.
      • Giedroc D.P.
      Sulfide homeostasis and nitroxyl intersect via formation of reactive sulfur species in Staphylococcus aureus.