Microbial Pathways in Colonic Sulfur Metabolism and Links with Health and Disease

REVIEW ARTICLE published: 28 November 2012 doi: 10.3389/fphys.2012.00448 Microbial pathways in colonic sulfur metabolism and links with health and disease

Franck Carbonero 1, Ann C. Beneﬁel 1, Amir H. Alizadeh-Ghamsari 1 and H. Rex Gaskins 1,2,3,4,5*

1 Department of Animal Sciences, University of Illinois, Urbana, IL, USA 2 Division of Nutritional Sciences, University of Illinois, Urbana, IL, USA 3 Department of Pathobiology, University of Illinois, Urbana, IL, USA 4 Institute for Genomic Biology, University of Illinois, Urbana, IL, USA 5 University of Illinois Cancer Center, University of Illinois, Urbana, IL, USA

Edited by: Sulfur is both crucial to life and a potential threat to health. While colonic sulfur metabolism Stephen O’Keefe, University of mediated by eukaryotic cells is relatively well studied, much less is known about sulfur Pittsburgh Medical Center, USA metabolism within gastrointestinal microbes. Sulfated compounds in the colon are either Reviewed by: of inorganic (e.g., sulfates, sulfites) or organic (e.g., dietary amino acids and host mucins) Shobna Bhatia, Seth G S Medical College and K E M Hospital, India origin. The most extensively studied of the microbes involved in colonic sulfur metabolism Jiyao Wang, Fudan University, China are the sulfate-reducing bacteria (SRB), which are common colonic inhabitants. Many *Correspondence: other microbial pathways are likely to shape colonic sulfur metabolism as well as the H. Rex Gaskins, Laboratory of composition and availability of sulfated compounds, and these interactions need to be Mucosal Biology, University of examined in more detail. Hydrogen sulfide is the sulfur derivative that has attracted the Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, most attention in the context of colonic health, and the extent to which it is detrimental IL 61801, USA. or beneficial remains in debate. Several lines of evidence point to SRB or exogenous e-mail: [email protected] hydrogen sulfide as potential players in the etiology of intestinal disorders, inflammatory bowel diseases (IBDs) and colorectal cancer in particular. Generation of hydrogen sulfide via pathways other than dissimilatory sulfate reduction may be as, or more, important than those involving the SRB. We suggest here that a novel axis of research is to assess the effects of hydrogen sulfide in shaping colonic microbiome structure. Clearly, in-depth characterization of the microbial pathways involved in colonic sulfur metabolism is necessary for a better understanding of its contribution to colonic disorders and development of therapeutic strategies.

Keywords: sulfur, hydrogen sulﬁde, colonic microbiota, sulfate-reducing bacteria, inﬂammatory bowel disease, colorectal cancer, irritable bowel syndrome

INTRODUCTION influence of microbial sulfur metabolism in the human intestine The crucial role of the microbiome in digestive processes and than previously recognized. intestinal health is now fully integrated in gastroenterology The sulfur present in the human body is provided exclusively research (Gordon et al., 2007).Theimmensenumberofmicrobes by diet and either converted to sulfated compounds, assimi- resident in the small intestine and colon form complex commu- lated by host cells or excreted. Sulfur is generally acquired in nities and metabolic interactions, which impact the host ben- the human diet through protein (Stipanuk, 2004). It is note- eficially or detrimentally. Accordingly, the intestinal microbiota worthy that one essential and another conditionally essential plays a central role in digestive biochemistry and element cycling. amino acid (methionine and cysteine, respectively) are sulfated, Whereas abundant data are available regarding microbial carbon and thus, sulfur acquisition is crucial to humans. Cysteine is metabolism (carbohydrate or fiber degradation), less is known considered a conditionally essential amino acid as it can be about microbial metabolism of other elements in the human synthesized from methionine via transsulfuration (Dominy and intestine. Stipanuk, 2004). As reviewed elsewhere, in addition to dietary Historically, the sulfur cycle was one of the first to be well doc- inputs, host sulfur amino acids are actively recycled through a umented in environmental microbiology (Kertesz, 2000; Canfield wide array of metabolic pathways (Stipanuk, 1986, 2004; Stipanuk and Farquhar, 2012). Chemolithoautotrophic and metabolic con- and Dominy, 2006). Similar to the host, gut microbes require version of sulfur compounds were first described in the late 19th sulfur inputs and, because of their active metabolism and tremen- century by Beijerinck (Kelly et al., 1997) and are of central interest dous number, are likely to play a major role in the metabolism in the context of environmental pollution (Kellogg et al., 1972). of luminal sulfur. Consequently, gut microbes are key players in Many sulfur compounds are also recognized for their toxicity determining the balance of beneficial to detrimental effects of and impact on human health (Ware et al., 1981). While there is sulfur-containing compounds (Table 1). This review summarizes no evidence for a microbial sulfur cycle per se in digestive sys- microbial pathways influencing sulfur metabolism and their rec- tems, direct and indirect evidence points to a greater diversity and ognized and potential contributions to colonic health and disease.

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Table 1 | Microbial taxa involved in pathways of colonic sulfur metabolism*.

Sulfur source Sulfur-containing substrate Microbial genus References

Inorganic Sulfate (SO42−) Desulfovibrio spp. (Desulfomonas spp) Gibson et al., 1988; Fite et al., 2004; Marchesi et al., 2009; Nava et al., 2012 Desulfobacter spp. Gibson et al., 1988; Nava et al., 2012 Desulfobulbus spp. Gibson et al., 1988; Nava et al., 2012 Desulfotomaculum spp. Gibson et al., 1988; Nava et al., 2012 Sulﬁte (SO32−) Bilophila wadsworthia Baron et al., 1989 Campylobacter jejuni Kelly and Myers, 2005 Organic Cysteine Escherichia coli Metaxas and Delwiche, 1955; Shatalin et al., 2011 Staphylococcus aureus Shatalin et al., 2011 Salmonella thyphimurium Kredich et al., 1972 Mycobacterium tuberculosis Wheeler et al., 2005 Helicobacter pylori Kim et al., 2006 Leishmania major (protozoa) Nowicki et al., 2010 Prevotella intermedia Igarashi et al., 2009 Fusobacterium nucleatum Yoshida et al., 2010 Streptococcus anginosus Yoshida et al., 2011 Clostridium spp. Genomic cysteine desulfhydrase Enterobacter spp. Genomic cysteine desulfhydrase Klebsiella spp. Genomic cysteine desulfhydrase Desulfovibrio spp. Genomic cysteine desulfhydrase Sulfomucin Prevotella strain RS2 Roberton et al., 2000 Bacteroides fragilis Roberton et al., 2000 Helicobacter pylori Slomiany et al., 1992 Akkermansia spp. Genomic glycosulfatase Taurine Bilophila wadsworthia Laue et al., 1997 Sulfated bile acids (rat model) Clostridium perfringens Huijghebaert and Eyssen, 1982; Huijghebaert et al., 1982; Robben et al., 1986 Estrogen-3-sulfates and phenylsulfates Peptococcus niger Vaneldere et al., 1991

*Assimilatory sulfate reduction, widespread and present in virtually all microbes, has not been included.

INTESTINAL SOURCES OF SULFUR Many other trace sulfated compounds are provided by dietary Food sources of inorganic sulfate include commercial breads, elements. In particular, fermented foodstuffs contain a wide array dried fruits, vegetables, nuts, fermented beverages, and bras- of volatile sulfur compounds (Landaud et al., 2008). Microbial sica vegetables (Florin et al., 1991). Diets supplemented with and host cell metabolism also produce a large variety of simple inorganic sulfate stimulate hydrogen sulfide (H2S) production to complex sulfated compounds available for further microbial within the colon (Christl et al., 1992; Lewis and Cochrane, 2007). utilization or degradation. Furthermore, in vitro incubation studies using human feces indicate that organic sulfur-containing compounds including cys- MICROBIAL PATHWAYS INVOLVED IN COLONIC SULFUR teine, taurocholic acid, and mucin provide a more efficient source METABOLISM for sulfide production than inorganic sulfate (Florin, 1991; Levine ASSIMILATORY SULFATE REDUCTION et al., 1998), with meat being a particularly important source Among microbes, assimilatory sulfate reduction is the most (Magee et al., 2000). In particular red meat, eggs, and milk con- widespread and essential biochemical process linked to sul- tain elevated concentrations of cysteine. Concentrations of both fur and provides a mechanism by which microorganisms can free cysteine and methionine in colon are relatively low (Ahlman reduce sulfate to elemental sulfur in order to satisfy phys- et al., 1993), indicating efficient metabolism by the microbiota iological requirements (Peck, 1961). This process is medi- and host epithelial cells. An additional source of colonic sul- ated by phosphoadenosine-5 -phosphosulfate (PAPS) reductases fur includes the sulfomucins. Mucins, consisting of a peptide (Figure 1), which are widely distributed among microbes and backbone, are largely responsible for the viscous properties of other living organisms except animals (for example 2924 hits for the colonic mucus layer and can be broadly classified as sialo- annotated nucleotide sequences and 10,402 for protein sequences mucins or sulfomucins based on the presence of terminal sialic were found in the NCBI database). Assimilatory sulfate reduction acid or sulfate groups, respectively, on the oligosaccharide chain can also be performed by other enzymatic pathways (Vaneldere (Verdugo, 1990; Jass and Roberton, 1994; Croix et al., 2011). et al., 1991; Seitz and Leadbetter, 1995) and likely represents

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FIGURE 2 | Dissimilatory sulfate reduction pathway. Microbial sulfate reduction relies on sequential catalytic reactions in which reduction of sulfate is coupled with oxidation of H2 or simple organic molecules. This anaerobic respiration pathway is less favorable thermodynamically than aerobic respiration.

describing SRB diversity in various environments. SRB have thus far been detected and quantified from stool and colonic mucosa FIGURE 1 | Overview of the microbial pathways involved in colonic samples (Zinkevich and Beech, 2000; Fite et al., 2004; Nava et al., sulfur metabolism. The sulfate remaining from assimilatory sulfate 2012). The genus Desulfovibrio has generally been found in higher reduction is available for sulfate-reducing bacteria and, thus, H2S production. Taurine and cysteine are additional potentially important numbers (Fite et al., 2004; Marchesi et al., 2009), with lower abun- substrates for microbial production of H2S. Various microbial metabolic dances of Desulfobacter, Desulfobulbus, and Desulfotomaculum pathways influence the composition and relative abundance of organic (Nava et al., 2012). The biochemistry of dissimilatory sulfate sulfur compounds. reduction has been investigated most extensively within species of Desulfovibrio. In the environment, SRB are able to utilize a wide range of substrates as electron donors and acceptors and can even a crucial step in sulfur cycling as it allows for significant dis- adopt non-sulfidogenic lifestyles (Plugge et al., 2011). For exam- posal of sulfate and conversion to necessary organic sulfated ple Desulfobulbus spp. can ferment organic matter under certain compounds. conditions (Kuever et al., 2005). It is not known if colonic SRB possess the ability to switch from sulfidogenic to non-sulfidogenic DISSIMILATORY SULFATE REDUCTION lifestyles in the colon, but an ability to adapt to varying sulfate While the process of assimilatory sulfate reduction is widespread levels would confer a competitive advantage. among microbes, only restricted microbial groups are capable of dissimilatory sulfate reduction (Figure 2). The sulfate- CYSTEINE DEGRADATION reducing bacteria (SRB) are notable as the only microbes in Cysteine degradation to H2S mediated by L-cysteine desulfhy- intestinal ecosystems that rely on inorganic sulfate for con- drase (Figure 3) was described in the 1950s in the Escherichia coli servation of energy. This sulfate respiration pathway has been model, a well-known inhabitant of the human gut (Metaxas and conserved in five bacterial and two archaeal phyla (Wagner Delwiche, 1955). Since that time, L- or D-cysteine desulfhydrase et al., 1998). Most of the SRB belong to the Deltaproteobacteria have been described in diverse taxa (Kumagai et al., 1975)includ- with more than 25 genera, followed by the Gram-positive SRB ing a few intestinal pathogens such as Salmonella thyphimurium within the Clostridia (Desulfotomaculum, Desulfosporosinus, and (Kredich et al., 1972), Mycobacterium tuberculosis (Wheeler et al., Desulfosporomusa genera). The SRB use sulfate as a terminal elec- 2005), H. pylori (Kim et al., 2006), and the protozoan Leishmania tron acceptor for respiration, with the concomitant production major (Nowicki et al., 2010). L- or D-cysteine desulfhydrase have of H2S(Peck, 1961). In the colon, sulfate reduction is generally also been studied in various oral microorganisms as a source of associated with dihydrogen oxidation (Carbonero et al., 2012), malodor and abscess formation (Igarashi et al., 2009; Yoshida but the electrons may also be provided from the oxidation of et al., 2010, 2011). Those genera found in the oral cavity, namely organic compounds, such as lactate (Flint et al., 2009). SRB Streptococcus, Prevotella and Fusobacterium, are also common are ubiquitously present in the human intestinal mucosa (Nava inhabitants of the human colon. A search in the GenBank and EBI et al., 2012) and have been enumerated by cultivation-dependent databases revealed that cysteine desulfhydrase-encoding genes methods from human stool in numbers ranging from 103 to have been annotated in several common colonic genera such 1011/g (Leclerc et al., 1980; Gibson et al., 1988). In a culture- as Clostridium, Enterobacter, Klebsiella, Streptococcus,and,sur- based study by Gibson et al., the principal SRB were lactate- and prisingly, the SRB genus Desulfovibrio. Although desulfhydrase hydrogen-utilizing Desulfovibrio spp. (64–81%), acetate-utilizing activity has not been quantified in intestinal ecosystems and Desulfobacter spp. (9–16%), propionate- and hydrogen-utilizing the prevalence of the gene among intestinal taxa remains unde- Desulfobulbus spp. (5–8%), lactate-utilizing Desulfomonas spp fined, it appears likely that desulfhydrase-harboring microbes are (reclassified within the genus Desulfovibrio) (3–10%), and important producers of H2S in the human colon. acetate- and butyrate-utilizing Desulfotomaculum spp. (2%). Other microbial enzymes possess cysteine desulfhydrase activ- However, these observations are based on cultivation meth- ity, and, more generally, the ability to convert cysteine or cysteine ods, which underestimate true bacterial diversity. More recently analogs is widespread. 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FIGURE 4 | The taurine degradation pathway of Bilophila wadsworthia. Bilophila wadsworthia is the only known intestinal microbe that uses taurine as an electron acceptor for anaerobic respiration. The first two enzymatic reactions result in sulfite production. Sulfite is subsequently converted to H2S by a dissimilatory sulfite reductase that differs structurally from those used by sulfate-reducing bacteria.

FIGURE 3 | Pathways of cysteine degradation to H2S. Cysteine desulfhydrase is a key enzyme for initial microbial cysteine fermentation and pyruvate production. However, recent evidence indicates that three species has attracted much attention because of its links with var- other enzymes orthologous to eukaryotic enzymes may catalyze similar ious disease symptoms (Finegold et al., 1992; Arzese et al., 1997; reactions. The generation of H2S via these four enzymatic pathways may Aucher et al., 1998; Nakayama et al., 2000; Devkota et al., 2012). It surpass that by dissimilatory sulfate reduction, especially in hosts reduces sulfite after an initial degradation of taurine and harbors consuming a protein-rich diet. a different dissimilatory sulfite reductase than other SRB (Laue et al., 2001)(Figure 4). A sulfite oxidation metabolic pathway environmental archaea (Tchong et al., 2005). Beta C-S lyases or has also been demonstrated in the food-borne human pathogen cystathionases mediates the cleavage of cystathionine to homo- Campylobacter jejuni (Kelly and Myers, 2005); however, the exis- cysteine in E. coli (Zdych et al., 1995)andLactobacillus spp. tence of similar pathways in intestinal commensal microbes has (De Angelis et al., 2002; Irmler et al., 2008). Another example is not been described. the SRB Desulfovibrio desulfuricans, which is able to obtain sul- Taurine, a sulfated compound secreted by the eukaryotic host fate from cysteine degradation (Forsberg, 1980). Conversely, the that is crucial for chelating bile acids in addition to contributing prevalent O-acetyl-L-serine sulfhydrylase (OASS) and homocys- to osmoregulation, cardiac function, and retinal development is teine synthase mediate the microbial synthesis of cysteine from degraded by B. wadsworthia using a taurine:pyruvate aminotrans- O-acetyl-L-serine and sulfide in bacteria and archaea (Borup and ferase to obtain sulfite (Laue et al., 1997; Laue and Cook, 2000) Ferry, 2000; Tai and Cook, 2001). (Figure 4). An intriguing link between dietary fat and the utiliza- A recent paper provided evidence that a high proportion tion of taurochloric acid as a source of taurine for B. wadsworthia of bacterial genomes harbor orthologs of mammalian cys- is suggested by two recent studies (Swann et al., 2011; Devkota tathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and et al., 2012). Compared to mice fed a low fat or high polyun- 3-mercaptopyruvate sulfurtransferase (3MST), three enzymes saturated fat diet, mice fed a high milk fat diet demonstrated involved in H2Sproduction(Shatalin et al., 2011). The an overrepresentation of B. wadsworthia, which was determined authors confirmed that pathogenic strains, including E. coli and to be mediated by taurine-conjugated bile acids (Devkota et al., Staphylococcus aureus, converted cysteine to H2Sthroughactiv- 2012). Furthermore, milk fat was essential for colonization by B. ities of one of those enzymes. Thus, it may be hypothesized wadsworthia in the germ-free mouse colon. Swann et al. (2011) that many gut microbes are capable of cysteine conversion to demonstrated that the lack of an established microbiota results in H2S. Further, the authors demonstrated that sulfide produc- a dominance of taurine-conjugated bile acids over unconjugated tion reduces oxidative stress, thereby increasing antibiotic resis- or glycine-conjugated bile acids, suggesting that bile acids, whose tance potential (Shatalin et al., 2011). Indeed, those microbial composition is modulated by the gut microbiota, may serve as metabolic pathways are very poorly documented in gut commen- signaling molecules in liver and other tissues (Swann et al., 2011). sals, and characterization of these enzymes and associated genes Although taurine has also been demonstrated to be a substrate will be crucial to understanding if microbial-generated sulfide for SRB in marine microbial mats (Visscher et al., 1999), it is not plays a role in shaping the colonic ecosystem. known if colonic SRB are capable of taurine utilization.

TAURINE AND SULFITE RESPIRATION ARYLSULFATASES Only a few genera are able to gain energy through sulﬁte respi- Bile acids can be sulfated by the action of host sulfotrans- ration rather than direct sulfate respiration (Pukall et al., 1999; ferases, which allow their subsequent detoxiﬁcation and disposal Soulimane et al., 2011). Among those, the only intestinal resident (Alnouti, 2009). It was demonstrated in a rat model that the characterized to date is Bilophila wadsworthia, a close relative opposite conversion, bile acid desulfation, can be mediated by of Desulfovibrio spp. (Baron et al., 1989). This unique bacterial microbial enzymes with arylsulfatase activity (Robben et al.,

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1988). Arylsulfatases were purified from Clostridium perfrin- given the persistent colonization of the human colonic mucosa gens (Huijghebaert and Eyssen, 1982; Huijghebaert et al., 1982; by SRB (Nava et al., 2012). The first step in the sulfide oxidation Robben et al., 1986) inhabiting the rat intestine and are also pathway is catalyzed by sulfide quinone reductase (SQR), a widespread in several abundant colon genera. Little arylsulfatase mitochondrial flavoprotein that oxidizes H2Stoprotein-bound activity was reported in human stool (McBain and Macfarlane, persulfide. Subsequently, a sulfur dioxygenase and a sulfur 1998), which does not preclude a more extensive activity of transferase are thought to sequentially convert SQR-bound microbial arylsulfatase in the colonic ecosystem where signifi- persulfide into sulfite and thiosulfate, respectively (Wilson et al., cant concentrations of sulfated bile acids are found (Palmer, 1967; 2008; Kabil and Banerjee, 2010). Given that most of the colonic Hofmann et al., 2008). disorders for which a potential pathogenic role for sulfide exists are effectively gene-environment disorders, it is straight-forward GLYCOSULFATASES to envision how common polymorphisms in host genes encoding Some colonic microbes have evolved to degrade colonic mucins components of the sulfide oxidation pathway might underlie (Roberton and Stanley, 1982; Derrien et al., 2004, 2008). In ineffective epithelial sulfide detoxification and thereby predispose particular, microbial glycosulfatases catalyze the release of certain individuals to chronic sulfide-generated inflammation or sulfate from sulfomucins (Corfield et al., 1992; Roberton and genotoxicity. Wright, 1997). Glycosulfatase enzymes have been described primarily in the intestinal resident Prevotella strain RS2 and H2S as an endogenous signaling molecule Bacteroides fragilis (Roberton et al., 2000), but also in the Despite its potential inflammatory, toxic and genotoxic prop- stomach resident Helicobacter pylori (Slomiany et al., 1992). A erties, there is also evidence that human cells may be able to suite of glycosulfatase-like enzymes were also described in the use H2S as a inorganic substrate for mitochondrial energiza- intestinal Peptococcus niger, using less abundant substrates such tion (Bouillaud et al., 2007). It is postulated that H2Smaybe as estrogen-3-sulfates and phenylsulfates (Vaneldere et al., 1991). an important endogenous signaling molecule (Abe and Kimura, Relative abundance and activity of glycosulfatase-harboring 1996; Distrutti et al., 2006). In addition, H2S has been sug- bacteria may greatly influence colonic sulfur metabolism; how- gested to be an effective drug against various GI diseases in ever, little is known about prevalence, abundance, and activity a rodent model (Wallace et al., 2007, 2009; Wallace, 2010). of bacterial glycosulfatases. A search in the GenBank and EBI However, the potential therapeutic properties of exogenous H2S databases confirmed that glycosulfatases encoding genes have still remain controversial (Bouillaud and Blachier, 2011; Olson, been annotated in important colonic genera such as Prevotella, 2011). Intriguingly, a recent report demonstrated that inhibi- Bacteroides (Arumugam et al., 2011), and Akkermansia (Belzer tion of H2S synthesis in healthy rats resulted in mucosal injury and De Vos, 2012). and inflammation in the small intestine and colon, while intra- colonic administration of H2S donors significantly reduced the LINKS WITH COLONIC HEALTH AND DISEASE severity of trinitrobenzene sulfonic acid-induced colitis (Wallace HYDROGEN SULFIDE et al., 2009). These data indicate that the outright assumption Toxicity that colonic H2S is only deleterious may be challenged and jus- tify additional study of both bacterial and endogenous sources of Hydrogen sulfide is the most suspect sulfated compound in the H S in the human colon. etiology of colonic disorders and inflammation (Nakamura et al., 2

2010; Danese and Fava, 2011; Medani et al., 2011). In addition, H2S as an architect of microbiome structure it is among the most hazardous gases in industrial applications, In the context of culture-based microbiology, sulfide in differ- exhibiting toxicity to different organs at low concentrations, with ent forms has been used as a strong reducing agent, allowing increasing concentrations being fatal (Guidotti, 1996). There maintenance of anoxia in media. However, sulfide concentra- is ample evidence that H2S at physiological concentrations is tions must be kept low to avoid toxicity in microbial cells. For genotoxic and induces DNA damage as well as inflammatory example, in microcosms simulating anaerobic digesters, lactose responses (Attene-Ramos et al., 2006, 2007, 2010). Exogenous fermentation and methanogenesis were inhibited by sulfide in a hydrogen sulfide is highly toxic to colonocytes and impairs their dose-dependent manner. Interestingly, it was also observed that metabolic function, especially butyrate oxidation (Roediger et al., SRB activities were inhibited by increased sulfide concentrations 1993; Babidge et al., 1998). In the human colon, sulfide exists (Hilton and Oleszkiewicz, 1988). In surface sediments of sandy largely in the volatile, highly toxic undissociated form (H2S), intertidal flats, high sulfide concentrations may reduce functional which is quickly absorbed by the mucosa or passes as flatus diversity (Freitag et al., 2003). Finally, sulfide production was (Suarez et al., 1997). Over 90% of sulfate disappears during pas- shown to provide cytoprotection from oxidative stress (Shatalin sage through the colon of individuals lacking a sulfate-reducing et al., 2011). As a start, in vitro studies with representative colonic microbiota, indicating that additional colonic processes compete strains are needed to better understand the potential for sulfide as for sulfate (Strocchi et al., 1993). well as sulfated compounds to influence the structure of colonic ecosystem. Detoxification Hydrogen sulfide is oxidized in many tissues including colonic INFLAMMATORY BOWEL DISEASE mucosa (Bartholomew et al., 1980; Furne et al., 2001), which The two major forms of inflammatory bowel disease (IBD), presumably is exposed frequently to bacterial-derived sulfide, Crohn’s disease (CD), and ulcerative colitis (UC), afflict 0.1–0.5%

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of individuals in Western countries (Podolsky, 2002; Hanauer, (Duffy et al., 2002). Finally, in a later study, the severity of pou- 2006). It is commonly accepted that IBD results from multi- chitis was positively correlated with fecal concentrations of H2S factorial interactions among genetic and environmental factors (Ohge et al., 2005), possibly reflecting a pathogenic role for this that lead to a dysregulation of the innate immune response to gas. Coffey et al. (2009) proposed that the surgical creation of the intestinal microbiota in genetically predisposed individuals ileo-anal pouches in UC patients might lead to colonic meta- (Podolsky, 2002). plasia, which, in turn, could result in increased production of Substantial evidence exists for a potential pathogenic role sulfomucin and, thus, higher colonization by SRB. The adverse of H2SinIBD,particularlyinUC(Pitcher and Cummings, consequences of such colonization would be greater exposure to 1996). Healthy colonic epithelial cells depend on the availabil- potentially proinflammatory concentrations of H2S(Coffey et al., ity of short-chain fatty acids such as butyrate for nutrition. 2009). Butyrate is produced during colonic fermentation and oxidized Increased activity of mucin sulfatase, an enzyme belonging by colonocytes via the enzyme acyl-CoA dehydrogenase. Because to the glycosulfatase group, was observed in patients with active this enzyme is inhibited by H2S, oxidation of butyrate is impaired UC but not CD (Tsai et al., 1995). In most patients, fluctuations by H2S(Babidge et al., 1998). Patients with UC were shown to in fecal sulfatase activity corresponded with severity of clini- have reduced breath excretion of CO2 after administration of cal disease, suggesting that the increased fecal sulfatase activity butyrate intrarectally, reflecting reduced colonic butyrate oxida- contributed to perpetuation of the disease. In this scenario, indi- tion (Den Hond et al., 1998). UC patients with reduced colonic viduals genetically predisposed to a high SRB carriage rate who butyrate oxidation also exhibit increased intestinal permeabil- then experience increased sulfatase activity might be at increased ity (Den Hond et al., 1998). Indeed, as ion absorption, mucin risk of UC due to the increased availability of endogenous sul- synthesis, membrane lipid synthesis, and detoxification processes fate for SRB sulfide production. Similarly, diets high in exogenous in colonocytes depend on the oxidation of butyrate (Roediger sources of sulfate would represent the greatest risk for those et al., 1997), decreases of butyrate oxidation would be expected genetically predisposed to a higher abundance of SRB. to compromise epithelial barrier function (Ramakrishna et al., Recently, a culture-dependent study demonstrated that 1991). mucosal biopsies from IBD patients were more often colonized Patients with UC have been shown to consume more pro- by Fusobacterium spp. than those from matched healthy con- tein and, thereby, more sulfur amino acids than control subjects trols (Strauss et al., 2011). It was further demonstrated that (Tragnone et al., 1995). Removing foods such as milk, cheese, Fusobacterium isolates from IBD patients trigger inflammatory and eggs improved symptoms in UC patients (Truelove, 1961). pathways in colonic cells (Dharmani et al., 2011). Fusobacterium Further, the numbers of SRB and rate of sulfidogenesis were spp. produce H2S through cysteine desulfhydrase activity. It also reported greater in UC patients than control cases (Gibson et al., appears possible that direct consumption of cysteine can reduce 1991; Pitcher et al., 2000). In a study comparing patients with in some instances sulfate availability for SRB, thereby possibly IBD to healthy individuals or patients with other gastrointestinal explaining the conflicting reports of SRB association with IBD. symptoms, the prevalence of Desulfovibrio piger detected via PCR The recent demonstration of selection for B. wadsworthia in the was significantly higher in the IBD patients (Loubinoux et al., gutmicrobiotaofmicefedahigh-saturatedfatdietthatwas 2002). Levine et al. (1998) found that production of H2Sfrom associated with higher occurrence and severity of colitis com- feces of patients with UC was 3–4 times greater than from feces paredtothosefedhigh-unsaturatedfat(Devkota et al., 2012) of healthy controls. However, this difference in H2Sproduction provides a potential mechanistic model for the development of may not have been due to colonization by a greater number of IBD in susceptible individuals. Notably, this effect was mediated SRB, as qPCR did not show that patients with active UC harbored by an increase of specific bile acids, particularly taurocholic acid, more SRB in stool or rectal mucosa than healthy controls (Fite a potential source of taurine for B. wadsworthia. et al., 2004). A common treatment regimen for patients with UC may contribute to conflicting results regarding the density of SRB COLORECTAL CANCER populations in UC patients. Specifically, 5-aminosalicylic acid (5- Colorectal cancer is the third most frequent cancer worldwide and ASA), an anti-inflammatory medication commonly prescribed the fourth most common cause of cancer death, with respon- for UC, also inhibits SRB growth and production of H2S(Pitcher sibility for 4,92,000 deaths annually (Weitz et al., 2005; Ferlay et al., 2000; Edmond et al., 2003). For example, stool sulfide con- et al., 2010). Genetic and environmental factors play a signifi- centrations between patients with UC and non-colitic controls cant role in the development of colorectal cancer (Kinzler and did not differ when the use of salicylates in colitic patients was Vogelstein, 1996; Rhodes and Campbell, 2002; de la Chapelle, not controlled (Moore et al., 1998); in those patients with UC 2004). Although etiologically divided into sporadic (90% of the who were not administered 5-ASA, fecal sulfide concentrations cases), hereditary (5–10%), and IBD-associated (2%), all colorec- were significantly greater (Pitcher et al., 2000). tal cancers show multistep development with several mutations Additional evidence for the role of H2SinUCistheobser- (Kinzler and Vogelstein, 1996; Rhodes and Campbell, 2002; de la vation that SRB were found in surgically constructed ileo-anal Chapelle, 2004). Doll and Peto (1981) estimated that over 90% pouches of UC patients but not in pouches of patients with famil- of gastrointestinal cancers are determined by environmental fac- ial adenomatous polyposis (FAP). Furthermore, H2Sproduction tors such as diet. It has been suggested that environmental cancer in UC pouches was 10 times greater than that in FAP pouches risk is determined by the interaction between diet and colonic

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microbial metabolism (O’Keefe et al., 2007). Particularly, there is abnormal bowel habits (Mayer, 2008). Diarrhea or constipation strong epidemiologic and experimental evidence that diets with may predominate or these symptoms may alternate. Microbial high animal fat and protein (meat) are associated with increased dysbioses have been described, and as microbial gases excreted in risk of colorectal cancer (Willett et al., 1990; Sandhu et al., 2001; breath are higher in IBS patients, abnormal microbial pathways Norat et al., 2002). As discussed earlier, meat contains rela- are a possible cause (King et al., 1998). tively high dietary sulfur, which can promote microbial sulfate Consistent with this possibility, it was demonstrated recently reductioninthecolon. that exogenous H2S (NaHS) inhibits in vitro motor patterns in Kanazawa and colleagues (1996)demonstratedthatH2Scon- the human, rat and mouse colon and jejunum mainly through centrations were significantly greater in patients who had pre- an action on multiple potassium channels (Gallego et al., 2008). viously undergone surgery for sigmoid colon cancer and who Exogenous H2S (NaHS) inhibited nociception induced by col- later developed new epithelial neoplasia of the colon, compared orectal distension in both healthy and post-colitic rats (Distrutti to individuals of similar age with a healthy colon. The abil- et al., 2006) but, conversely, triggered visceral nociceptive behav- ity of the colon to detoxify H2S is also reduced in patients ior when administered intracolonically in the mouse (Matsunami with colon cancer (Ramasamy et al., 2006). The association of et al., 2009). Recently, a functional dysbiosis was described in H2S with colon cancer is further supported by the finding that constipation-predominant IBS patients with, in particular, a sig- H2S induces colonic mucosal hyperproliferation, with this effect nificant increase of SRB and fecal sulfide, indicating a potential reversed by butyrate (Christl et al., 1996). The mucosal hyper- role for sulfide in the development of IBS symptoms (Chassard proliferation effect of H2S may be mediated by mitogen-activated et al., 2012). protein kinase (MAPK)-mediated proliferation (Deplancke and Gaskins, 2003). Hydrogen sulfide is also a potent genotoxin that CONCLUSIONS induces direct radical-associated DNA damage (Attene-Ramos Microbial sulfur metabolism in the human colon is likely more et al., 2006, 2007).ColoncancerinUCand,perhaps,spo- extensive than has been previously recognized. For example, H2S, radic colon cancer in general may reflect genomic instability the sulfated compound with the highest potential influence on resulting from exposure to H2S(Attene-Ramos et al., 2007). As digestive health, can be generated from cysteine degradation as the number of SRB was reported to be not significantly differ- well as via dissimilatory sulfate reduction. The extent to which ent (Balamurugan et al., 2008) or reduced in colorectal cancer organic sulfur metabolism is operative in the human colon, patients when compared to healthy controls (Scanlan et al., 2009), how diet influences relative activities of the enzymatic pathways impaired detoxification of H2S may be critical to the role of this involved, and which organisms carry out these processes are compound in colon cancer. poorly understood. In addition, the importance of the microbiota Increased detection of Fusobacterium spp. in CRC tumors has and the metabolism of sulfated bile acids are now established, been reported in two independent studies (Kostic et al., 2011; and further work is needed to understand how dietary fat intake Castellarin et al., 2012). As described previously, Fusobacterium influences these pathways. With clear evidence that the human spp. produce H2S through cysteine desulfhydrase activity. As H2S colonic mucosa is persistently colonized by SRB, the beneficial has been demonstrated to be genotoxic, it can be hypothesized versus toxic effects of H2S need to be delineated. One possibil- that it may be one causative metabolite in the etiology of CRC. ity is that the mucosal microbiome is shaped in part through It also appears possible that direct bacterial consumption of cys- the differential susceptibility of mutualistic microbes to sulfide teine would limit sulfate availability for SRB, thereby possibly toxicity. Clearly, extensive work is needed to understand the pri- explaining the conflicting reports of SRB association with CRC. mary pathways for the production of H2S and how their activities may influence both host and microbial components of the colonic IRRITABLE BOWEL SYNDROME ecosystem. This new body of knowledge may identify relatively Irritable bowel syndrome (IBS) is a functional bowel disor- innocuous approaches for the prevention or treatment of chronic der characterized by chronic abdominal pain, bloating, and colonic disorders.

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abscess formation in BALB/c mice. is an enzyme with the activity of commercial or financial relationships This article was submitted to Frontiers in Mol. Oral Microbiol. 26, 221–227. a beta-C-S lyase (cystathionase). that could be construed as a potential Gastrointestinal Sciences, a specialty of Yoshida, Y., Ito, S., Kamo, M., Kezuka, J. Bacteriol. 177, 5035–5039. conflict of interest. Frontiers in Physiology. Y., Tamura, H., Kunimatsu, K., et al. Zinkevich, V., and Beech, I. B. (2000). Copyright © 2012 Carbonero, Benefiel, (2010). Production of hydrogen Screening of sulfate-reducing bac- Received: 10 August 2012; accepted: 08 Alizadeh-Ghamsari and Gaskins. This is sulfide by two enzymes associated teria in colonoscopy samples from November 2012; published online: 28 an open-access article distributed under with biosynthesis of homocysteine healthy and colitic human gut November 2012. the terms of the Creative Commons and lanthionine in Fusobacterium mucosa. FEMS Microbiol. Ecol. 34, Citation: Carbonero F, Benefiel AC, Attribution License,whichpermitsuse, nucleatum subsp nucleatum 147–155. Alizadeh-Ghamsari AH and Gaskins HR distribution and reproduction in other ATCC 25586. Microbiology 156, (2012) Microbial pathways in colonic forums, provided the original authors 2260–2269. Conflict of Interest Statement: The sulfur metabolism and links with health and source are credited and subject to any Zdych,E.,Peist,R.,Reidl,J.,andBoos, authors declare that the research and disease. Front. Physio. 3:448. doi: copyright notices concerning any third- W. (1995). MalY of Escherichia coli was conducted in the absence of any 10.3389/fphys.2012.00448 party graphics etc.

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