Microbial Assimilatory Sulfate Reduction-Mediated H2S: An Overlooked Role in Crohn's Disease Development

Microbial Assimilatory Sulfate Reduction-Mediated H2S: An Overlooked Role in Crohn's Disease Development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Microbial Assimilatory Sulfate Reduction-Mediated H 2 S: An Overlooked Role in Crohn's Disease Development Wanrong Luo, Min Zhao, Mohammed Dwidar, Liyuan Xiang, Yang Gao, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4176488/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Aug, 2024 Read the published version in Microbiome → Version 1 posted 4 You are reading this latest preprint version Abstract Background H 2 S imbalances in the intestinal tract trigger Crohn's disease (CD), a chronic inflammatory gastrointestinal disorder characterized by microbiota dysbiosis and barrier dysfunction. However, a comprehensive understanding of H 2 S generation in the gut, and the contributions of both microbiota and host to systemic H 2 S levels in CD, remain to be elucidated. This investigation aimed to enhance comprehension regarding the sulfidogenic potential of both the human host and the gut microbiota. Results Our analysis of a treatment-naive CD cohorts' fecal metagenomic and biopsy metatranscriptomic data revealed reduced expression of host endogenous H 2 S generation genes alongside increased abundance of microbial exogenous H 2 S production genes in correlation with CD. While prior studies focused on microbial H 2 S production via dissimilatory sulfite reductases, our metagenomic analysis suggests the assimilatory sulfate reduction (ASR) pathway is a more significant contributor in the human gut, given its high prevalence and abundance. Subsequently, we validated our hypothesis experimentally by generating ASR-deficient E. coli mutants ∆cysJ and ∆cysM through the deletion of sulfite reductase and L-cysteine synthase genes. This alteration significantly affected bacterial sulfidogenic capacity, colon epithelial cell viability, and colonic mucin sulfation, ultimately leading to colitis in murine model. Further study revealed that gut microbiota degrade sulfopolysaccharides and assimilate sulfate to produce H 2 S via the ASR pathway, highlighting the role of sulfopolysaccharides in colitis and cautioning against their use as food additives. Conclusions Our study significantly advances understanding of microbial sulfur metabolism in the human gut, elucidating the complex interplay between diet, gut microbiota, and host sulfur metabolism. We highlight the microbial ASR pathway as an overlooked endogenous H 2 S producer and a potential therapeutic target for managing CD. Inflammatory bowel disease inorganic sulfate sulfopolysaccharide 3’-Phosphoadenosine-5’-phosphosulfate PAPS Adenosine-5’-phosphosulfate APS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Crohn's disease (CD) and ulcerative colitis (UC) are two main forms of Inflammatory Bowel Disease (IBD), characterized by symptoms including diarrhea, rectal bleeding, abdominal pain, fatigue, and weight loss, significantly impacting patients' lives. IBD incidence and prevalence are rising globally, particularly in newly industrialized regions 1 , 2 . The growing global burden of this disease underscores the need for preventive and therapeutic measures 2 . Although the precise etiology remains elusive, it is believed to result from dysregulated mucosal immune responses triggered by gut bacteria, especially in individuals with genetic predispositions 3 , 4 . Sulfur metabolism and sulfur-containing metabolites play a pivotal role in IBD 5 – 7 . Hydrogen sulfide (H 2 S) is the sulfur derivative that garners the most attention in the context of colonic health. In the gastrointestinal system, the H 2 S pathway supports epithelial, immune, and enteric nervous system health through various mechanisms, including posttranslational modification of protein cysteine residues, activation of K ATP channels, and serving as an inorganic fuel for colonocytes 8 – 10 . However, excessive exposure to H 2 S can be detrimental to the host, damaging the intestinal epithelium and leading to chronic inflammation, as well as disrupting the balance between cellular proliferation and apoptosis 11 . An association between elevated H 2 S levels and IBD has long been suspected 12 , 13 . Several studies suggest that pharmacological interventions targeting H 2 S may improve outcomes in IBD through mechanisms such as driving regulatory T cell differentiation, stabilizing hypoxia-inducible factor 1-alpha (HIF-1α), promoting biofilm formation, and reducing planktonic bacteria growth 14 – 16 . However, a comprehensive mechanistic model elucidating the relationship between H 2 S generation and IBD is still lacking. The production and release of H 2 S are regulated by both endogenous and exogenous factors, but the relative contributions of the host and gut microbiota to overall systemic H 2 S levels in humans remain uncertain. Endogenous H 2 S production primarily results from the enzymatic degradation of organic sulfur compounds, particularly cysteine. Key enzymes in this process include cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH), 3-mercaptopyruvate sulfur transferase (MPST), and methanethiol oxidase (SELENBP1). On the other hand, our understanding of microbial-mediated H 2 S generation remains limited 17 , 18 . Bacteria produce H 2 S through the utilization of both organic sulfur compounds like L-cysteine and taurine, as well as inorganic sulfur compounds such as sulfate and sulfite. The two primary pathways for sulfate metabolism are Assimilatory Sulfate Reduction (ASR), involving the reduction of sulfate to H 2 S, which is subsequently incorporated into cysteine and methionine biosynthesis, and Dissimilatory Sulfate Reduction (DSR), a process found in sulfate-reducing bacteria where these microbes produce H 2 S from sulfate without integrating it into L-cysteine (Fig. 1 ). Previous investigations into microbial sulfidogenesis found the contributions of gut microbiota to systemic total H 2 S levels varied widely across subjects 19 , and have mainly focused on the fermentation of organic sulfur compounds 5 , 20 and sulfate-reducing bacteria DSR 21 , 22 . Meanwhile, ASR, a common strategy employed by many microbes to fix sulfur and manipulate organosulfur compounds, has been routinely overlooked. Here, we employed genomic and metagenomic tools to gain a deeper understanding of the colonic sulfidogenic capacity of both the host and gut microbiome in a newly-onset treatment-naïve CD cohort, and observed that CD exhibit reduced endogenous H 2 S production alongside increased gut microbial H 2 S generation, primarily via the ASR pathway. Mechanistically, we genetically manipulated E. coli ASR pathway to evaluate the impact on (i) E. coli 's sulfidogenic capacity, (ii) colon epithelial cell viability, and (iii) the development of colitis and maintenance of mucus integrity in a mouse model. Our data elucidate a previously unappreciated role of microbial ASR pathway in dietary sulfate metabolism, intestinal sulfur homeostasis and mucus integrity, emphasizing its pivotal role in CD pathogenesis. Results CD is associated with up-regulation of gut microbial assimilatory sulfate reduction We conducted a comprehensive investigation into the H 2 S production capabilities of the human gut microbiome, focusing on key genes responsible for sulfide generation from various sources, including organic compounds (such as dietary rich L-cysteine and taurine) and inorganic sulfate (Fig. 1 , Supplementary dataset 1). Our analysis was based on stool metagenomic samples from two independent IBD cohorts, FAH-SYSU (treatment naïve IBD cohort enrolled at the First Affiliated Hospital of Sun Yat-sen University) 23 and PRISM (Prospective Registry of IBD study at MGH) 3 . ShortBRED was employed to identify unique sequence markers of related family members and quantifying their relative abundance in metagenomic data with high specificity 24 . It is noteworthy that ShortBRED was not specifically developed for gene cluster identification and quantification. Nevertheless, our search within metagenomic datasets using individual genes revealed a relatively high degree of consistency among genes from the same cluster (spearman r 0.59–0.94, p < 0.001, Supplementary dataset 2), confirming the accuracy of the predictions. We found that genes associated with assimilatory sulfate reduction (ASR), including sulfate adenylyltransferase (CysND) and adenylylsulfate kinase (CysC), were prevalent (100% and 80–100% in FAH-SYUS and PRISM cohort, respectively) and abundant (98.0-646.7 and 20.6–57.2 RPKM in FAH-SYSU and PRISM cohort, respectively, Fig. 2 ) in both CD patients and HC control subjects, although the PRISM cohort exhibited lower abundance, possibly due to differences in sequencing procedures. In the ASR pathway, organisms use different strategies: 1) Adenosine-5’-phosphosulfate (APS) is phosphorylated into 3’-Phosphoadenosine-5’-phosphosulfate (PAPS) by CysC, which is further reduced into sulfite (SO 3 2− ) by PAPS reductase (CysH); 2) APS is directly reduced by an APS reductase (AprAB) to generate adenosine monophosphate (AMP) and SO 3 2− . Both scenarios generate SO 3 2− which could further be reduced by anaerobic sulfite reductase (AsrABC) or sulfite reductase (CysJI) to form sulfide (S 2− ), which subsequently yield L-cysteine mediated by cysteine synthase A (CysK ) and cysteine synthase B (CysM) (Fig. 1 ). We found ASR downstream genes, including cysH , cysJI , cysM , cysK , and asrABC , were also more abundant in CD subjects ( p < 0.01), suggesting a significant role for ASR in H 2 S production from SO 4 2− in CD individuals (Fig. 2 A, Supplementary dataset 3). We observed that the prevalence and abundance of dsrAB genes (key genes for the DSR but not the ASR pathway) were notably lower compared to asr -associated genes in both cohorts. In the PRISM cohort, dsrAB genes were detected in approximately 30.9–32.4% of CD subjects, while this percentage increased to 67.6–76.5% in HC subjects. Moreover, their abundance increased from approximately 0.48 to 0.60–0.72 RPKM ( p < 0.01). However, no significant difference in dsrAB genes was observed in the FAH-SYSU cohort (Fig. 2 A). Additionally, aprAB , responsible for converting APS to SO 3 2− in both DSR and ASR, showed a marked reduction in CD subjects (Fig. 2 A). Furthermore, we investigated cysPUWA , which encodes a sulfate transporter common to both pathways. These transporter genes were more prevalent and abundant in CD subjects, indicating increased microbial sulfate transport in CD patients. Organic sulfur metabolism has been reported to be enriched in individuals with IBD and colorectal cancer (CRC) 25 , 26 . Therefore, we investigated microbial genes associated with organic sulfur metabolism in our study. We found that the bacterial gene mpst , crucial for converting L-cysteine to H 2 S, exhibited more prevalent and significantly elevated levels in CD subjects compared to HC subjects in both cohorts ( p < 0.001, Fig. 2 A). Additionally, genes related to taurine and alkanesulfonate metabolism, including taurine transporter ( tauABC ), taurine dioxygenase ( tauD ), sulfonate transporter ( ssuACB ), and alkanesulfonate monooxygenase ( ssuD ), were more abundant in CD subjects in FAH-SYSU cohort ( p < 0.001, Fig. 2 A). Microbial methanethiol oxidase ( mtoX ), widely distributed in the biosphere 27 , was not detected in human associated bacteria, and therefore was excluded in the ShortBRED analysis. In the PRISM cohort, an increasing trend was observed in tauABC and ssuD among CD subjects, although statistical significance was not attained. CD patients demonstrate impaired endogenous H 2 S production Endogenous H 2 S production arises from the host's utilization of sulfur-containing amino acids (Fig. 1 ). To shed light on endogenous sulfidogenic activity, we evaluated the expression levels of the host cbs , cth , mpst and selenbp1 by examining intestinal biopsies obtained from newly diagnosed CD patients (n = 46) and non-disease controls (n = 44) from the FAH-SYSU cohort 28 . Our analysis revealed that all of these 4 genes exhibited significant decreases in inflamed mucosal biopsies from CD subjects (Fig. 2 B). A similar trend was observed in three independent IBD cohorts, including the Mount Sinai Hospital cohort (GSE83687) 29 , a treatment-naive pediatric IBD cohort (E-MTAB-5464) 30 and the HMP IBD cohort 4 (Fig. 2 B), although statistical significance was not achieved in some cases. In the E-MTAB-5464 cohort, transcriptomic data were generated from purified intestinal epithelial cells. CBS raw counts in this cohort were generally less than 10, hence not analysed. This finding strongly suggests a substantial reduction in the endogenous sulfidogenic capacity of CD patients. To uncover whether CD patients have impaired H 2 S detoxification capacity, we examined the expression levels of key enzymes responsible for host H 2 S detoxification in these cohort datasets, including thiosulfate sulfurtransferase (TST), thiosulfate sulfide:quinone oxidoreductase (SQOR), and persulfide dioxygenase (ETHE1) (Fig. S1 A) 13 , 31 . Our analysis revealed that tst was significantly downregulated, while sqor was upregulated in CD subjects in FAH-SYSU cohort. A similar trend was observed in the HPM IBD cohort, although tst did not reach statistical significance between CD and non-IBD groups in this cohort. The expression of ethe1 remained similar in both FAH-SYSU and HMP cohorts, irrespective of CD or control groups (Fig. S1 B). Therefore, further research is needed to explore the H 2 S detoxification capacity in CD patients. Oxygen-insensitive ASR is functionally more active in fecal microbiota from CD patients To further substantiate the contribution of the ASR pathway from gut microbiota to H 2 S generation in CD, we conducted an ex vivo fecal culture experiment using thiosulfate (S 2 O 3 2 − 2− ) as the sole sulfur source. DSR has been reported in sulfate-reducing bacteria which are strictly anaerobes, whereas ASR has been reported in facultative anaerobes and aerobes 32 . Therefore, we set up the fecal culture aerobically and measured H 2 S production in fecal samples from both healthy individuals and CD patients to test if oxygen-insensitive ASR activity was enhanced in CD’s gut microbiota. We detected H 2 S production in 19 out of 34 (55.9%) CD stool samples, with 13 showing notably high levels (> 10,000 intensity). In contrast, only 6 out of 35 (17.1%) samples from healthy controls exhibited H 2 S production, with 5 demonstrating high levels (Fig. 2 C). Thus, oxygen-insensitive ARS is more active in CD patients. The bacterial ASR pathway is prevalent in the human microbiome The asr gene cluster in E. coli MG1655 33 and Salmonella enterica ST8493 34 , along with the dsr gene cluster in Desulfovibrio gigas DSM 1382 35 , that have been characterized in previous studies, are shown in Fig. 3 A. To comprehensively assess the distribution of asr - ( cysDN , cysC , cysH , cysJI , cysM, cysK , aprAB and asrABC ) and dsr -associated genes ( dsrAB , aprAB ) among human bacteria, we screened these genes against 1635 Human Microbiome Project (HMP) reference genomes. This extensive analysis revealed that asr -associated genes are more widespread than dsrAB (Fig. 3 B, Supplementary dataset 4). A significant number (88.1%, 1441 out of 1635 reference genomes) of the total reference genomes contain at least one asr -associated gene, distributed predominantly in Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes, whilst dsrAB genes are only found in 0.43% (7 genomes) which are from Firmicutes and γ-Proteobacteria (Fig. 3 B, Supplementary dataset 4). The prevalence of cysDN (409 genomes) and cysC (295 genomes) in Bacteroidetes were higher than the other asr -associated genes. On the other hand, cysJI (193 strains) and cysM (275 strains) were more prevalent in Proteobacteria, including facultative aerobic species like E. coli , Proteus. mirabilis , Klebsiella. oxytoca (Fig. 3 B, Supplementary dataset 4). asrABC (117 genomes) was more commonly found in Firmicutes and Fusobacteria. These observations suggest a potential collaborative interplay among microorganisms in the execution of the ASR pathway. Construction of E. coli mutants with impaired assimilatory sulfate reduction (ASR) We focused on the cysJI -mediated ASR pathway in this study since metagenomic data indicated that it is more abundant than asrABC (Fig. 2 A). We used E. coli MG1655, a known bacterium with a complete ASR pathway, as the model organism (Fig. 4 A). Using homologous recombination, we deleted two crucial ASR pathway genes, cysJ and cysM . cysJ encodes sulfite reductase alpha subunit ( cysI encodes beta subunit), responsible for the reduction of SO 3 2 − 2− to S 2 − 2− while cysM encodes cysteine synthase B, which converts S 2− to L-cysteine. (Fig. 4 A). As expected, deleting cysJ hindered E. coli growth on SO 4 2− as the sole sulfur source (Fig. 4 B and Fig. S2 A). E. coli carries a CysM homologue, CysK, which compensates for CysM in incorporating S 2− into L-cysteine (Fig. 4 A). Consequently, E. coli ∆cysM strains exhibited growth similar to the WT strain when SO 4 2− was the sole sulfur source. Alternatively, CysM can use thiosulfate (S 2 O 3 2− ) in place of S 2− to produce L-cysteine via S-sulfocysteine as the intermediate (Fig. 4 A). Therefore, E. coli WT and ∆cysJ mutant grew on S 2 O 3 2− as the sole sulfur source, however ∆cysM displayed diminished growth rate (Fig. 4 B and Fig. S2 B). We assessed E. coli WT and mutant strains for their sulfidogenic capabilities using various inorganic and organic sulfur sources. In the modified Sulfur, Indole, Motility (SIM) medium, we observed that deleting cysJ increased H 2 S production from L-cysteine, while deleting cysM enhanced H 2 S generation from both L-cysteine and SO 3 2− (Fig. 4 C). E. coli WT also produced H 2 S from L-cysteine, as indicated by slight medium darkening (Fig. 4 C). We further cultured E. coli WT and mutant strains in M9 medium supplemented with varying concentrations of L-cysteine, and observed that all the strains exhibited a dose-dependent production of H 2 S, with the ∆cysJ mutant demonstrating greater efficiency in converting L-cysteine to sulfide than ∆cysM and WT (Fig. 4 D). The enhanced H 2 S productivity of the ∆cysJ mutant remained consistent under anaerobic condition (Fig. S2 C). Additionally, the ∆cysJ mutant accumulated SO 3 2− in M9 medium when SO 4 2− or S 2 O 3 2− was the sole sulfur source due to the loss of sulfite reductase activity (Fig. 4 E). These findings highlight the impact of ASR pathway alterations on both inorganic and organic sulfur metabolism. E. coli WT and ASR-deficient mutants displayed distinct morphological characteristics and proteomic profiles (Fig. S3A, B, Supplementary dataset 5), suggesting that the alteration of the ASR pathway has a profound effect on bacterial physiology. Bacterial assimilatory sulfate reduction modulates epithelial cell viability We proceeded to investigate the impact of modifications in the bacterial ASR pathway on the growth of colonic epithelial cells in an in vitro setting. We co-cultured E. coli WT and mutant strains with normal human colonic mucosal epithelial cell line NCM460 with either L-cysteine or S 2 O 3 2− as the sole sulfur source. Cell viability assay revealed that in the presence of L-cysteine, the ∆cysJ mutant led to significantly decreased cell viability, concurrent with increased H 2 S production (Fig. 4 D, F). When S 2 O 3 2− served as the exclusive sulfur source, the ∆cysM mutant induced more pronounced cell death, accompanied by higher H 2 S generation (Fig. 4 C, F). As ∆cysJ mutant accumulates more SO 3 2− in the medium when SO 4 2− is the sole sulfur source (Fig. 4 E), this accumulation of SO 3 2− may potentially leads to cell toxicity 36 . To investigate it further, we collected the supernatants from cultures of E. coli WT and mutant strains grown in M9 medium supplemented with Na 2 SO 4 and used them to treat NCM460 cells (Fig. 4 G). As indicated by the cell proliferation assay, the ∆cysJ mutant exhibited the most pronounced inhibition of cell proliferation in agreement with the high levels of SO 3 2− (Fig. 4 H). Thus, the data suggest that bacterial ASR modulates epithelial cell viability through SO 4 2− metabolites. The gut microbiota is the primary contributor to serum H 2 S levels in the DSS-induced mouse colitis model Colitis, a key component of IBD, is frequently studied using murine models. One widely employed method to induce colitis in these models is the administration of dextran sodium sulfate (DSS) via drinking water. Our initial objective was to determine if H 2 S production is linked to the DSS-induced colitis model. We found a significant increase in serum H 2 S levels in mice received DSS compared to vehicle controls (Fig. 5 A), suggesting that serum H 2 S is associated with DSS-induced colitis. Although a previous study suggested that germ-free mice exhibit reduced plasma H 2 S levels 37 , the specific contribution of the gut microbiota to systemic H 2 S levels in the context of DSS-induced colitis remained unknown. To illuminate the link between elevated serum H 2 S and gut microbes, we performed two studies. First, we utilized a publicly available colonic tissue transcriptomic dataset from mice undergoing DSS-induced colitis, followed by a tissue regeneration phase (GSE131032) 38 . During the colitis and recovery stages, the expression of cbs and cth genes remained stable, while mpst and selenbp1 expression displayed a decreasing trend during colitis, followed by a slight elevation during the recovery stage (Fig. 5 B). This suggested that endogenous H 2 S production remained consistent or even decreased during DSS-induced colitis, hence the rise in serum H 2 S observed is probably from gut microbiota. Second, we administered broad spectrum antibiotics (Abx) to mice in the DSS-induced model (Fig. 5 C), and observed a significant reduction in serum H 2 S levels and alleviated DSS-induced colitis, as evidenced by weight and colon length measurements (Fig. 5 D-G). Given mRNA levels of cbs and cth remained stable throughout the DSS-induced colitis and recovery stages (Fig. 5 B), we further examined protein levels of CBS and CTH in Abx-challenge mice experiment, and observed no significant difference between the two groups (Fig. 5 H). Collectively, these findings provide compelling evidence that the gut microbiota plays a central role in the elevation of systemic H 2 S levels in the DSS-induced colitis model. Therefore, we utilized this model to investigate the causal relationship between microbial ASR pathway and colitis in vivo . The gut bacterial ASR pathway contributes to sulfide generation derived from dietary sulfate Diet plays a pivotal role in shaping the composition and metabolic activity of the gut microbiota. While prior research mainly concentrated on organic sulfur compounds from dietary proteins, the role of inorganic sulfur (SO 4 2− ) remains understudied 20 , 39 . Carrageenan, a common sulfated polysaccharide food additive, is linked to UC relapse risk and can induce intestinal inflammation in animal model 40 , 41 . We hypothesized that gut microbiota-mediated carrageenan degradation and subsequent H 2 S production might contribute to its pro-colitis effects. To test this hypothesis, we initially cultured E. coli WT and mutant strains in M9 medium supplemented with λ-carrageenan as the sole sulfur source, owing to its high sulfur content (32–39%, Fig. 6 A). Surprisingly, both WT and mutant strains demonstrated H 2 S production (Fig. 6 A), which contrasted with previous findings that ∆cysJ mutant couldn’t grow on inorganic SO 4 2− . Given that carrageenan is a biopolymer derived from red algae, it likely contains trace amounts of organic sulfur compounds that can be utilized by ∆cysJ mutant. As a result, we transitioned to DSS, a synthetic sulfated polysaccharide with approximately 18–20% sulfur content (Fig. 6 B), which as mentioned earlier, is a commonly used as inducer in murine colitis models 42 . We first tested whether the gut microbiota was involved in DSS degradation. Abx-treated mice exhibited significantly higher fecal DSS levels compared to vehicle control mice, suggesting active DSS degradation by gut microbiota in vivo (Fig. 5 C, 6 C). Ex vivo experiments with mouse and human stool samples showed about 35% of the DSS was consumed after overnight incubation (Fig. 6 D), confirmed microbiota-mediated DSS degradation. We postulated that DSS degradation releases SO 4 2− , which are subsequently assimilated by bacteria employing the ASR pathway. To test this hypothesis, we initiated an experiment involving 1% DSS incubation with human/mouse fecal cultures for 16 hours, followed by supernatant collection, and subsequent inoculation with E. coli WT and mutant strains (Fig. 6 E). As anticipated, E. coli WT and mutant strains formed H 2 S in the presence of pre-incubated DSS. E. coli ∆cysM mutant produced higher levels of H 2 S than WT and the ∆cysJ mutant (Fig. 6 E). Proteus mirabilis , which carries the asr -gene cluster, generated H 2 S as well (Fig. 6 E, Supplementary dataset 4). Direct culture of E. coli ∆cysJ mutant in M9 medium with DSS as the sole sulfur source did not yield growth (Fig. 6 F). Although E. coli WT and ∆cysM mutant grew on DSS as the sole sulfur source, they showed low utilization and negligible DSS degradation, emphasizing metabolic cross-feeding among bacterial species for efficient sulfated polysaccharide metabolism (Fig. 6 F-G). The ASR pathway modulates DSS-induced colitis in vivo SPF mice received Abx-cocktail were subsequently inoculated with E. coli WT, ∆cysJ and ∆cysM , then subjected to DSS administration (Fig. 7 A). Mice colonized with the E. coli ∆cysJ mutant exhibited elevated serum H 2 S and fecal SO 3 2− levels associated with more severe disease phenotype evidenced by a greater body weight loss, a worsening of disease activity, and more severe intestinal inflammation characterized by increased mucosal erosion, crypt destruction and inflammatory cell infiltration in the colon (Fig. 7 B-H). The heightened serum H 2 S levels may be ascribed to the degradation of organic sulfur compounds within the gastrointestinal tract, such as L-cysteine, catalyzed by E. coli ∆cysJ . We found a significant reduction in plasma levels of ursodeoxycholic acid (UDCA), α- and ω-muricholic acid (MCA), and an increase in cholic acid-7-sulfate (CA-7S) in mice colonized with the ∆cysJ strain (Fig. S4A), indicating that alteration of E. coli ASR pathway strongly influenced the bile acid profile in mice. While ∆cysM and WT-colonized mice exhibited similar disease severity, serum H 2 S and fecal SO 3 2− levels, a significant difference in colonic mucin composition was observed. The colonic mucus layer, essential for maintaining homeostasis between resident microbiota and underlying immune cells, is primarily composed of acidomucins, broadly categorized as sialomucins or sulfomucins depending on the presence of sialic acid or sulfate groups 43 . Colonic tissues from ∆cysM -colonized mice showed a reduced sulfomucin:sialomucin ratio in (Fig. 6 I-K), indicating compromised host sulfation than WT mice. Intestinal sulfation, crucial for colitis protection, is dependent on the host PAPS synthase 2 (PAPSS2), which is central in generating PAPS, the universal sulfonate donor for sulfation 36 . Our analysis of transcriptomic data from the FAH-SYSU, HMP and GSE83687 cohorts indicated a significant decrease in colonic papss2 gene expression in actively inflamed CD patients compared to non-disease and non-IBD controls (Fig. 7 L). Mice deficient in papss2 have been previously demonstrated to manifest reduced intestinal sulfomucin content, rendering them susceptible to DSS-induced colitis 36 . Therefore, we reasoned that reduced host sulfate in ∆cysM -colonized mice were due to the downregulation of papss2 . Indeed, we observed decreased colonic mRNA expression of papss2 in both ∆cysJ and ∆cysM groups compared to the WT group, as confirmed by real-time PCR analysis (Fig. 7 M). Discussion The burden of IBD, which encompasses conditions like CD and UC, is substantial and often leads to hospitalizations and surgical interventions 1 . Current treatments primarily target host inflammatory pathways using non-specific immunosuppressive agents, which can pose significant risks and may not always be effective, necessitating the exploration of alternative approaches 44 . Mounting evidence suggests that an imbalance in H 2 S production, either insufficient or excessive, can act as an environmental trigger for CD 22 , 45 . Studies have shown that the administration of H 2 S donors can suppress the expression of proinflammatory cytokines and ameliorate colitis in murine models 12 , 46 . This raises the possibility that modulating H 2 S concentrations in the gut lumen could be an exciting therapeutic strategy for treating CD 47 . However, research into this potential link between H 2 S and CD has been hindered by a limited understanding of sulfur metabolism within the human gut. To address this knowledge gap, we conducted a comprehensive investigation into the functional capacity of both the gut microbiota and host in H 2 S production. Our findings suggest that microbial sulfur metabolism within the human colon is more complex and widespread than previously recognized. We analyzed metagenomic data from independent IBD cohorts and found that CD is associated with an increase in microbial generation through ASR pathways, as evidenced by the increased abundance and prevalence of asr -associated genes. Ex vivo fecal culture confirmed ASR-mediated H 2 S generation is more functionally active in stool samples from CD patients. Using E. coli as the model organism, we generated ∆cysJ and ∆cysM mutants deficient in the ASR pathway. We conducted in vitro and in vivo studies to validate that the bacterial ASR pathway modulates cell viability, host sulfate homeostasis, and colitis pathogenesis. Our investigation has brought into focus the pivotal role played by ASR pathway in reshaping the utilization of L-cysteine and generation of H 2 S. The deletion of cysJ gene in E. coli amplifies H 2 S production from L-cysteine. The heightened metabolism of L-cysteine by gut microbes and increased abundance of cysM has recently been associated with CRC 26 . We noticed that asrABC is enriched in CD subjects, warranting further investigation into its potential association with CD. In contrast to previous research, our findings suggest that the DSR pathway is unlikely to be the primary contributor to the elevated fecal microbial sulfidogenic capacity in CD. Prior research on exogenous H 2 S generation primarily centered on DSR, based on the culturing and sequencing of Desulfovibrio genus, sulfate-reducing bacteria frequently found in the human and animal gut 21 , 48 . However, Anantharaman et al. 49 revealed that dsrAB -mediated dissimilatory sulfur metabolism is predicted in a much broader diversity of bacterial and archaeal groups than previously recognized, primarily due to horizontal gene transfer, such as Bilophila wadsworthia , an opportunistic pathogen inhabiting the gut. Consequently, it is more reasonable to predict DSR-mediated H 2 S generation based on dsr -gene cluster quantification, rather than relying solely on Desulfovibrio quantification. Analysis of intestinal biopsy transcriptomic data from multiple IBD cohorts has unveiled a compromised endogenous sulfidogenic capacity in CD patients. This is evident from the downregulation of key genes, specifically cbs , cth , mpst and selenbp1 . Severe CD manifestations in a child with cbs deficiency has been reported 50 . Reduced expression of cbs mpst and selenbp1 has been linked to the exacerbation of inflammation-induced intestinal barrier injury in UC and CD 51 – 53 , . Animal studies have provided additional evidence highlighting the critical role of endogenous H 2 S generation in colitis. MPST −/− and MPST +/− mice exhibit exacerbated DSS-induced colitis 52 . Inhibition of endogenous H 2 S synthesis through the use of CBS and CTH inhibitors, such as β-cyanoalanine, propargylglycine, and O-carboxymethyl-hydroxylamine hemihydrochloride, has been demonstrated to worsen colitis in mouse model 12 . Further investigations are warranted to elucidate the factors responsible for the downregulation of genes involved in endogenous H 2 S generation. Considerable efforts are underway to investigate the potential contributions of dietary factors to the pathophysiology of IBD 54 , 55 . The interplay of genetic, environmental, microbial, and immunological factors makes diet a crucial aspect of IBD etiology 56 . Dietary sulfur intake, primarily from inorganic sulfate and sulfur-containing amino acids (SAAs) such as methionine, cysteine, and taurine, plays a significant role 5 , 20 . However, estimations of dietary sulfur content often fail to account for sulfur-containing food modifiers or additives, such as carrageenan and sulfiting agents ( e.g. , potassium bisulfate, sodium bisulfate) 57 . Daily intake of inorganic sulfate is estimated to range from 1.5 to 16.0 mmol 57 . Interestingly, fecal sulfate (and sulfide) excretion is minimal compared to dietary intake, suggesting that sulfate is actively removed from the fecal stream during passage through the gut by both host and gut microbiota 58 . Carrageenan, a sulfated polysaccharide, contains approximately 15–40% sulfur, depending on the specific type of carrageenan ( e.g. , kappa, iota, lambda) and the seaweed species used for extraction 59 . Carrageenan is widely used as a food additive in the Western diet, and its consumption has substantially increased over the past 50 years, paralleling the rising prevalence of IBD 60 . Successful dietary interventions that induced CD remission have excluded processed foods containing carrageenan, further supporting the notion that carrageenan may trigger or exacerbate inflammation in IBD 61 . In animal models, carrageenan administration consistently induces intestinal ulcerations resembling human IBD histopathologically 41 . We reasoned that SO 4 2− released during carrageenan degradation are utilized by gut microbes, contributing to IBD pathogenesis. To investigate, we used synthetic sulfated polysaccharide DSS due to carrageenan's organic sulfur contaminants. The DSS-induced colitis model is known for consistently mimicking epithelial damage seen in IBD, underscoring the complex interplay between dietary elements, gut microbiota, and disease pathogenesis 42 . DSS mouse colitis model is known for its variability even among genetically identical mice and across different mouse facilities. A recent study found that gut microbiota plays a significant role in driving this variability within the model 62 . Through in vivo and ex vivo studies, we've demonstrated that the gut microbiota can degrade DSS, releasing SO 4 2− that fuel bacterial ASR pathways. This alteration in microbial sulfur metabolism ultimately modulates disease severity. Our study highlights the crucial role of gut microbial ASR metabolism in dietary sulfate metabolism and susceptibility to colitis. It's important to acknowledge the significant daily intake of inorganic sulfate and the potential exacerbation of microbial H 2 S production by carrageenan used as food additives in processed foods, which can lead to mucosal damage. Furthermore, it is worth noting that CD pathogenesis is unlikely to be solely attributed to the presence and activities of single species. Genes associated with the ASR pathway exhibited varying distribution patterns among human bacteria. Additionally, in DSS degradation, mouse and human fecal microbial communities are more efficient than E. coli monocultures, suggesting cross-feeding among different bacterial species for the efficient metabolism of sulfated polysaccharides. In summary, our study reveals the extensive diversity of microbial sulfur metabolism pathways. These findings highlight the association between CD and reduced endogenous H 2 S production alongside increased gut microbial H 2 S generation, primarily via the ASR pathway. Microbial ASR-mediated dietary sulfate metabolism emerges as a crucial factor in colitis. Our research sheds light on the complex interaction between diet, the gut microbiota, and inorganic sulfate metabolism, highlighting their potential as promising therapeutic targets for managing CD. Materials and Methods Human subjects All study protocols abided by the Declaration of Helsinki principles and were approved by Ethical Committees of the First Affiliated Hospital of Sun Yat-sen University. Intestinal biopsies and stool specimens were collected as part of the FAH-SYSU cohort study (2016[113]). Subject stool samples were collected at the FAH, SYSU gastroenterology clinic and stored at -80°C immediately. For culturing assays, fecal samples were collected and diluted to make a 10% (w/v) fecal slurry by resuspension of the feces in 10% (w/v) glycerol solution, and aliquots were stored in cryogenic vials at -80°C until use. The exclusion criteria applied to all groups were as follows: recent (< 3 months prior) use of any antibiotic therapy, current extreme diet ( e.g. , parenteral nutrition or macrobiotic diet), known history of malignancy, current consumption of probiotics, any gastrointestinal tract surgery leaving permanent residua ( e.g. , gastrectomy, bariatric surgery, colectomy), or significant liver, renal, or peptic ulcer disease. Analyses of asr - and dsr -associated genes in Human Microbiome project (HMP) references genomes HMP references genomes (1635 genomes as of June 30, 2023) were selected and analyzed through the IMG program on the Joint Genome Institute website ( https://img.jgi.doe.gov/ ) 63 . The functions (Supplementary dataset 1) were used to carry out a “Function Profile” against all selected reference genomes to identify those carrying asr - and dsr -associated genes. Hits were manually inspected. Genomes carrying sulfidogenic gene(s) were selected to generate a phylogenetic tree using phyloT ( https://phylot.biobyte.de/ ) based on NCBI taxonomy and visualized using iTOL 64 . Genome and gene IMG ID are available in Supplementary dataset 4. Metagenomic data analysis We used ShortBRED 24 to accurately profile the abundance of genes involved in the H 2 S generation in metagenomes sourced from the FAH-SYSU (BioProject: PRJNA793776) 65 and PRISM (BioProject: PRJNA400072) 3 datasets. We initially compiled a set of identified bacterial sulfidogenic genes as our query sequences (Supplementary dataset 6). Subsequently, ShortBRED-Identify was employed to generate markers for these key bacterial sulfidogenic gene sequences using UniRef90 (May, 2023) as a reference list with an 85% cluster ID threshold. These markers were applied in ShortBRED-Quantify to assess gene abundance in paired metagenomes, which had previously undergone quality control via the KneadData workflow ( http://huttenhower.sph.harvard.edu/kneaddata ). The output from ShortBRED-Quantify was expressed as reads per million reads per kilobase million (RPKM). Cultivation of wild type bacteria and mutants Escherichia coli MG1655 wild type, mutants (Δ cysJ and Δ cysM ) and Proteus mirabilis ATCC 29906 were generally cultivated in Luria broth (LB) containing tryptone (10 g·l − 1 ), yeast extract (5 g·l − 1 ) and NaCl (10 g·l − 1 ). To characterize the growth of E. coli wild type and mutant strains, they were cultivated in 5 mL LB overnight at 37°C in a shaking incubator (250 rpm) and the pellet was collected by centrifugation at 3,000×g for 10 min. Cell pellets were then washed and re-suspended in fresh M9 media (inoculum size 1:20, v/v). The defined M9 medium contained NaCl (0.5 g·L − 1 ), KH 2 PO 4 (3 g·L − 1 ), Na 2 HPO 4 ·12H 2 O (6 g·L − 1 ), NH 4 Cl (1 g·L − 1 ), MgCl 2 (95 mg·L − 1 ), CaCl 2 (11.1 mg·L − 1 ) and glucose (0.1%, w/v). 1 mM Na 2 SO 4 , Na 2 S 2 O 3 , L-cysteine or DSS was used as sole sulfur source. 200 µL samples were collected from each tube at the indicated time points, and their optical density at 600 nm was measured in flat-bottom 96-well plates (200 µL per well). Sulfite in the supernatant was quantified as described in Sulfite Quantification section. Allele-exchange mutagenesis of ΔcysJ and ΔcysM in E. coli MG1655 DNA fragments (~ 1 kb) corresponding to the upstream and downstream regions of the target gene were amplified and a subsequent overlap PCR was used to fuse the two fragments which were then ligated into suicide plasmid harboring kanamycin resistance cassette, oriT (mob), sacB counter selection marker and R6K origin of replication using the In-Fusion HD Cloning kit (Clontech). The ligated suicidal plasmid (pKmobSac) was transformed into the donor strain, E. coli S17 λpir. In parallel, E. coli MG1655 was transformed with a temperature-sensitive ampicillin-resistance plasmid carrying oriR101 origin of replication (p101-Amp). The suicidal plasmid (pKmobSac) was then transformed into E. coli MG1655 through conjugation and the resulted conjugants were screened at room temperature on LB agar plates containing ampicillin at 100 ng/µL (to select against E. coli S17 donor cells) and kanamycin at 50 ng/µL. One single-crossover integrant was then selected and re-streaked on LSW-Sucrose agar plate (tryptone 10 g/L, yeast extract 5 g/L, glycerol 5 mL/L, NaCl 0.4 g/L, sucrose 100 g/L and agar 20 g/L) 66 supplemented with ampicillin at 100 ng/µL to select for the correct double cross-over mutants. One mutant was then selected, re-streaked, and confirmed for the loss of the conjugated plasmid through Sanger sequencing and its ability to grow in presence of ampicillin but not kanamycin. The knockout E. coli MG1655 mutants were then cured from p101-amp plasmid through growing at 37°C. H 2 S quantification Plasma H 2 S levels were quantified using a modified methylene blue method. In brief, ZnAC was added to 100 µL of plasma samples to precipitate H 2 S, HS − , S²-, and plasma proteins. Subsequently, the ZnS pellet was re-dissolved by adding 130 µL of 2% N, N-dimethyl-p-phenylenediamine and 130 µL of 20% trichloroacetic acid. Methylene blue formation was initiated by addition of FeCl 3 ·6H 2 O and quantified at 665 nm using a spectrophotometer. Microbial culture H 2 S levels were quantified by Modified S ulfur, I ndole, M otility (SIM)-medium and Lead Acetate Test Strip. See online supplemental material for further details. Dextran sulfate quantification To measure DSS concentration in mouse fecal pellets, Sample aliquots (50 µL) were injected onto a size exclusion column (SEC-150, 3 µM, 7.8 × 300 mm, Welch, Cat # 00237-21052) and eluted at a flow rate of 1.5 mL/min. The mobile phase consisted of 25 mM KH 2 PO 4 , 25 mM K 2 HPO 4 ·3H 2 O, 50 mM KCl, and 10% ethanol. The eluent passed through a post-column derivatization instrument (LABRAT, LYM-1060), where it mixed with a 10 µg/mL dimethylene blue zinc chloride double salt (DMB, Sigma, 34108) delivered directly by pump A connected to the online mixer. Detection was performed using a VWD detector at 530 nm wavelength, with data collected via OpenLAB CDS chromatography data software (Agilent, 1260 Infinity II). Sulfite quantification 150 µL of culture supernatant or caecum slurry extract was mixed with 350 µL distilled water and 10 µL 10 M NaOH. Sulfite was quantified using the Total Sulfite Assay Kit (JC-HX-04, HK) based on the Pararosaniline Method, following the manufacturer's instructions. The reaction formed a purple-red complex, pararosaniline methylsulfonic acid, which exhibited maximal absorption at 550 nm, and absorbance was measured after a 10-minute incubation using a plate reader (UV-2450, SHIMADZU, Japan). Cell culture and viability assay The NCM460 human colon epithelial cell line (RRID: CVCL_0460) was maintained in RPMI 1640 basic medium (gibco, 8123133) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 100 µg/mL penicillin G, and 100 µg/mL streptomycin sulfate (Invitrogen, Carlsbad, CA, USA). Cells were cultured at 37°C in a 5% CO 2 humidified incubator. Cell viability was analyzed by live/dead staining or Cell Counting Kit-8 (CCK8, GLPBIO, GK10001) according to the manufacturer's instructions. For live/dead staining, NCM460 cells (4 × 10 3 /well) in 96-well microplates were cultured for 48 hours in 100 µL of 1640 medium. After washing with PBS, cells were prepared for bacterial co-culture. E. coli strains were cultivated overnight at 37°C in LB, washed in sulfur-free M9 media, and resuspended in M9 medium with L-cysteine or Na 2 S 2 O 3 as the sole sulfur source. Cells were co-cultured with these bacteria for 2 hours (150 µL bacterial culture per well). Afterward, cells were stained with 30 µL calcein-AM/PI working solution (2 µM calcein-AM and 4.5 µM propidium iodide) at 37°C for 20 minutes, followed by fixation with 0.4% polyformaldehyde. Quadruplicate experiments were conducted, and images of cells were acquired and analyzed using an Olympus IX83 fluorescence microscope. All experiments were performed in quadruplicate. The images of the cells were acquired immediately and analyzed by using a fluorescence microscope (Olympus IX83). Positive cell percentages and average fluorescence intensity were determined using Image-Pro Plus 6.0. For CCK8 assay, NCM460 cells (5 × 10 3 /well) were cultured overnight in 96-well microplates and treated with 50 µL of bacterial supernatants from M9 medium cultures with 2 mM Na 2 SO 4 as the sole sulfur source. After a 6-hour incubation, cells were washed, incubated with 100 µL of RPMI 1640 Medium plus 10 µL CCK-8 reagent, and absorbance at 450 nm was monitored. All experiments were performed in six replicates and blank wells without cells served as controls. Animal Studies Male SPF C57BL/6 mice (6–8 weeks) were maintained on a standard normal rodent diet (Synergy Bio, AIN-93M). All the mice used in this study were bred and raised in the animal facility of the First Affiliated Hospital of Sun Yat-sen University. Mice (n = 6) received antibiotic cocktail (Abx) 67 for 5 days prior to 2% DSS ad libitum in drinking water for 6 days. Mice (n = 6) without Abx pre-treatment was used as vehicle control. Fecal samples from days 3 and 5 were processed by mixing 0.05 g of fresh feces with 500 µL PBS, vortexed for 3 min, and centrifuged at 14,000×g for 10 min. The supernatant was used for DSS quantification as described in DSS quantification section. For E. coli gavage experiment, mice received Abx-water were subsequently administered E. coli wild type and mutant strains via oral gavage at a dose of 1.0 × 10 9 cfu/200 µL each. DSS was supplied in drinking water at 3% (w/v) for 6 days. Caecum was harvested for sulfite quantification as described in Sulfite Quantification section. Mice were monitored daily for body weight, stool consistency and stool bleeding. Mice were euthanized by cervical dislocation after 5–6 days of DSS treatment. Colon samples were collected for histological, western blot and qRT-PCR analysis. Bacterial H 2 S Production from λ-Carrageenan and DSS Bacteria from the LB start culture were harvested, washed, and inoculated in 5 mL sulfur-free M9 medium with 1% λ-carrageenan (inoculum size 1:200, v/v ), incubated overnight at 37°C while a suspended lead acetate strip monitored H 2 S generation. Medium without inoculum was set up as a negative control. To assess DSS degradation, mouse and human fecal samples (2 CD subjects and 2 healthy individuals) were cultured in 10 mL LB medium (OD 600 = 1.0). Bacterial pellets were resuspended in 5 mL sulfur-free M9 medium with 1% DSS. DSS levels in supernatants were measured via SEC-HPLC, and H 2 S production was monitored with suspended lead acetate strips. To investigate H 2 S production from DSS by E. coli WT, mutants, and P. mirabilis , cultures were grown overnight in 10 mL LB medium at 37°C, 250 rpm. After harvesting, cells were resuspended in 1 mL sulfur-free M9 medium. Pre-incubated DSS medium was prepared using supernatant from M9 medium with 1% DSS and cultures from two CD subjects, as described earlier. Subsequently, 200 µL of this pre-incubated DSS medium and sulfur-free M9 medium with 1% DSS were inoculated with 50 µL of bacterial cultures. Incubation was conducted at 37°C in a 96-well plate with lead acetate strips for sulfide quantification (see Sulfide Quantification, Lead Acetate Strip section). Statistical Analysis Statistical analyses were performed with Prism v.8.0 (GraphPad). For two-group comparisons, the statistical significance was determined by unpaired t test or nonparametric Mann-Whitney test as indicated. Multiple group comparisons were made by ANOVA for most of the studies as indicated. Each data point denotes individual human subject, animal, or biological replicate. Declarations Ethics approval and consent to participate Study research protocols were reviewed and approved by the Ethical Committees of the First Affiliated Hospital of Sun Yat-sen University (2016[113]). Written informed consent was obtained from all participants. All animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the First Affiliated Hospital of Sun Yat-sen University (2021 [303], 2023 [183]). Consent for publication Not applicable. Funding This work is supported by the National Natural Science Foundation of China (82100577 to Y.Z., 82270579 to R. F., 82370551 to M. C.), Natural Science Foundation of Guangdong Province (2021A1515010572 to R.F.). Availability of data and materials All study data are included in the article and/or SI Appendix. Data are available in a public, open access repository. Gene expression profiling by high-throughput sequencing data have been deposited in Gene Expression Omnibus accession no. (GSE83687 and GSE131032) and Biostudies accession no. (E-MTAB-54674). Metagenomic sequences for the PRISM is available via SRA with BioProject number PRJNA400072. Raw metagenomic data of the FAH-SYS cohort were deposited in the NCBI public repository (Bioproject #PRJNA793776). HMP IBD metagenomics and transcriptomic data can be accessed at https://ibdmdb.org/tunnel/public/summary.html. All plasmids, bacterial mutant strains, and reagents generated in this study are available from the lead contact upon completing Material Transfer Agreement. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Competing interests The authors declare no competing interests. Authors' contributions M.C., M.D., R. F., and Y.Z. designed research; W.L., M.Z., W.X., R.F., S.X., X.L., L.X., X.L, and Y. 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Supplementary Files MicrobiomeSI032724.docx Supplementarydataset17.xlsx Cite Share Download PDF Status: Published Journal Publication published 16 Aug, 2024 Read the published version in Microbiome → Version 1 posted Editorial decision: Revision requested 11 Apr, 2024 Editor assigned by journal 11 Apr, 2024 Submission checks completed at journal 28 Mar, 2024 First submitted to journal 27 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4176488","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285164251,"identity":"055c014b-5c14-4aff-8982-85b23d8ac7ca","order_by":0,"name":"Wanrong Luo","email":"","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Wanrong","middleName":"","lastName":"Luo","suffix":""},{"id":285164252,"identity":"000e5bac-1a6e-4b67-bf62-9aad94936746","order_by":1,"name":"Min Zhao","email":"","orcid":"","institution":"Shenzhen No.3 People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Zhao","suffix":""},{"id":285164253,"identity":"2ec8e3cc-9f04-4f86-a40d-31f7feebd106","order_by":2,"name":"Mohammed Dwidar","email":"","orcid":"","institution":"Cleveland Clinic","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"","lastName":"Dwidar","suffix":""},{"id":285164254,"identity":"43e96f6c-cc3b-4aa4-8e6e-86351e1199e5","order_by":3,"name":"Liyuan Xiang","email":"","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Liyuan","middleName":"","lastName":"Xiang","suffix":""},{"id":285164255,"identity":"601eb80e-5c53-47ff-b20f-f59bbbcac156","order_by":4,"name":"Yang Gao","email":"","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Gao","suffix":""},{"id":285164256,"identity":"1dc7911e-a46c-4534-9602-9920807d6c6c","order_by":5,"name":"Xueting Wu","email":"","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Xueting","middleName":"","lastName":"Wu","suffix":""},{"id":285164257,"identity":"a3f2c8ed-4637-48e1-8592-9f72d064141b","order_by":6,"name":"Marnix H. Medema","email":"","orcid":"","institution":"Wageningen University","correspondingAuthor":false,"prefix":"","firstName":"Marnix","middleName":"H.","lastName":"Medema","suffix":""},{"id":285164258,"identity":"95e94a58-8e54-4a8e-922a-335b994dc547","order_by":7,"name":"Shu Xu","email":"","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Shu","middleName":"","lastName":"Xu","suffix":""},{"id":285164259,"identity":"e1923e0b-216d-423c-9159-814600513744","order_by":8,"name":"Xiaozhi Li","email":"","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Xiaozhi","middleName":"","lastName":"Li","suffix":""},{"id":285164260,"identity":"4f661f8e-eef4-4556-ab76-c586fdf7ad24","order_by":9,"name":"Hendrik Schaefer","email":"","orcid":"","institution":"University of Warwick","correspondingAuthor":false,"prefix":"","firstName":"Hendrik","middleName":"","lastName":"Schaefer","suffix":""},{"id":285164261,"identity":"07bedf31-3707-430d-ac0a-791ddad9e7b0","order_by":10,"name":"Minhu Chen","email":"","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Minhu","middleName":"","lastName":"Chen","suffix":""},{"id":285164262,"identity":"e2b7c39b-5f9d-4f4a-aee2-6de4ffb0f9cd","order_by":11,"name":"Rui Feng","email":"","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Feng","suffix":""},{"id":285164263,"identity":"7e2b4227-976a-478b-8541-16ab4a933e57","order_by":12,"name":"Yijun Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYDACCRCu+C/HwMDYeABZkICWM8zGQC0NJGhhbGFObADSxGmRn91jJmHZwJa+tv0w0JY/h+0NDjAfvM3DYJeHSwvjnDNmEpI7eHK3nUlsOMDYdjhxwwG2ZGsehuRiXFqYJXKAWs5I5G47ANLScDjB4ACPmTQPwwGwU7EBNrCWNoN0s/MPYQ7j/4ZXCw9ES0KC2Q2gLQxshxk3HOBhw6tFQiKt2ELizAHDbTeAtiS2pSfOPMxmbDnHIBmnFvkZyRtvS1QckDc7n/7wwYc/1vZ8x5sf3nhTYYdTCxCwSMOjIYGhGRgiIJYBbvVAwPzxA4JTh1fpKBgFo2AUjEwAAFx4Wx9dYiWGAAAAAElFTkSuQmCC","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":true,"prefix":"","firstName":"Yijun","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2024-03-27 13:35:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4176488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4176488/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40168-024-01873-2","type":"published","date":"2024-08-16T15:57:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53954698,"identity":"4ce384fa-c549-453e-bdfe-93e47fb4f007","added_by":"auto","created_at":"2024-04-02 16:43:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":272646,"visible":true,"origin":"","legend":"\u003cp\u003eExogenous microbial sulfur metabolism results in the production of genotoxic H\u003csub\u003e2\u003c/sub\u003eS \u003cem\u003evia\u003c/em\u003e metabolism of inorganic sulfate and organic sulfur like cysteine, taurine, isethionate, \u0026nbsp;methanethiol and alkanesulfonate (black and green). Endogenous H\u003csub\u003e2\u003c/sub\u003eS is produced \u003cem\u003evia\u003c/em\u003e metabolism of sulfur containing amino acids like cysteine and homocysteine, as well as organic sulfur methanethiol (green). Gene names and KEGG ID are listed in Supplementary dataset 1.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/59523070731dd639e69d93fc.png"},{"id":53954697,"identity":"c7595b52-e36e-4e80-9008-14c124b09ed0","added_by":"auto","created_at":"2024-04-02 16:43:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":357447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD is associated with decreased endogenous sulfidogenic gene expression and increased gut microbial exogenous sulfidogenic gene expression。\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Dot plots comparing selected genes related to microbial sulfide generation from CD versus healthy control subjects (HC) in FAH-SYSU (light green background) cohort and PRISM (light blue background) cohort. The size of each dot indicates the proportion of participants detected in each group of the indicated gene and the color of each dot indicates RPKM with that gene in each group. RPKM, reads per kilobase per million mapped reads. Genes related to ASR, DSR, and organic sulfur metabolism are color-coded according to the scheme in Figure 1.\u003c/p\u003e\n\u003cp\u003e(B) Analysis of \u003cem\u003ecbs\u003c/em\u003e, \u003cem\u003ecth\u003c/em\u003e, \u003cem\u003empst\u003c/em\u003e and \u003cem\u003eselenbp1\u003c/em\u003e gene expression in CD and non-IBD control subjects’ mucosa in different IBD cohorts. CBS, cystathionine beta-synthase; CTH, cystathionine gamma-lyase; MPST, 3-mercaptopyruvate sulfurtransferase; SELENBP1, methanethiol oxidase. * Various control groups were utilized in different cohorts. FAH-SYSU, non-disease control; HMP, symptomatic non-IBD controls; E-MTAB5464, non-disease control; GSE83687, normal non inflamed bowel away from the tumor from sporadic colon cancer patients.\u003c/p\u003e\n\u003cp\u003e(C) CD patients exhibit increased assimilatory sulfate reduction activity in their fecal microbial community compared to healthy subjects.\u003c/p\u003e\n\u003cp\u003eSignificance was determined by nonparametric Mann-Whitney test. *p \u0026lt;0.05, **p \u0026lt;0.01, ***p \u0026lt;0.001\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/19e81cd4b65a1e9c25a62276.png"},{"id":53954699,"identity":"5a7a8fd4-21eb-4b1e-8fc5-505611dd8e80","added_by":"auto","created_at":"2024-04-02 16:43:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":450998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASR pathway is widely distributed in human microbiota.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Arrangement of \u003cem\u003easr\u003c/em\u003e- and \u003cem\u003edsr\u003c/em\u003e- associated genes and gene clusters in bacteria. Orange ORFs: \u003cem\u003easr\u003c/em\u003e-associated genes; gray ORFs: \u003cem\u003edsr\u003c/em\u003e-associated genes. Other genes not shown are represented by white ORFs.\u003c/p\u003e\n\u003cp\u003e(B) Phylogenetic distribution of genomes harboing \u003cem\u003easr\u003c/em\u003e- and \u003cem\u003edsr\u003c/em\u003e-associated genes in 1635 reference genomes of the Human Microbiome Project (HMP). Gene and genome names are listed in Supplementary dataset 4.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/5ff229d94f93a6413fd328fd.png"},{"id":53954702,"identity":"e8f048a0-3a3f-483e-bc49-dd9fb78bd70f","added_by":"auto","created_at":"2024-04-02 16:43:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":796296,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeletion of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecysJ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecysM\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e alters \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS sulfidogenic capacities and modulate cell viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Scheme of the \u003cem\u003eE. coli\u003c/em\u003e MG1655 sulfate assimilation reduction pathway.\u003c/p\u003e\n\u003cp\u003e(B) Growth of \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains on M9 medium with 1 mM sulfate (left) or sodium thiosulfate (right) as the sole sulfur source.\u003c/p\u003e\n\u003cp\u003e(C) WT and mutant strains of \u003cem\u003eE. coli\u003c/em\u003e were qualitatively tested for H\u003csub\u003e2\u003c/sub\u003eS in SIM media of different sulfur sources, as evidenced by the formation of black FeS.\u003c/p\u003e\n\u003cp\u003e(D) Relatively quantitative test of H\u003csub\u003e2\u003c/sub\u003eS produced by \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains in M9 medium with different concentrations of L-cysteine as a sole sulfur source under aerobic conditions. Significance was measured with two-way ANOVA analysis with multiple comparisons.\u003c/p\u003e\n\u003cp\u003e(E) Quantification of sulfite (SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2−\u003c/sup\u003e) produced by \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains in M9 medium using 1mM Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e or Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as the sole sulfur source.\u003c/p\u003e\n\u003cp\u003e(F) Representative images and quantification of cell death rates cell death rate of NCM460 cells co-cultured with \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains, with either L-cysteine supplementation (upper panel) or sodium thiosulfate supplementation (lower panel), were analyzed using live/dead staining. Living cells are represented in green, while dead cells are shown in red.\u003c/p\u003e\n\u003cp\u003e(G-H) A CCK-8 assay was performed using NCM460 cells treated with supernatant from M9 medium containing 1 mM Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e that had been pre-inoculated with \u003cem\u003eE. coli\u003c/em\u003e WT or mutant strains.\u003c/p\u003e\n\u003cp\u003eMean ± SEM is displayed from at least three independent experiments. Significance was measured with one-way ANOVA analysis with multiple comparisons.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/34beb19e43be8f396acbb5f4.png"},{"id":53954700,"identity":"b8cab797-9685-446d-a826-c6b54db04d45","added_by":"auto","created_at":"2024-04-02 16:43:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":362944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDSS-induced colitis is associated with increased exogenous H2S generation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Serum H\u003csub\u003e2\u003c/sub\u003eS level of mice in normal control (NC) and DSS-treated group. Each dot represents an individual mouse. NC, mice were on water (n=6-7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(B)\u0026nbsp; The expression levels of \u003cem\u003ecbs\u003c/em\u003e, \u003cem\u003ecth\u003c/em\u003e, \u003cem\u003empst\u003c/em\u003e, and \u003cem\u003eselenbp1\u003c/em\u003e in murine colonic tissues were assessed during the administration of DSS and the subsequent recovery period (GSE131032, n=2−3). P-value was determined by nonparametric one-way ANOVA analysis with multiple comparisons. See Fig. 2 for gene full names.\u003c/p\u003e\n\u003cp\u003e(C) Schematic diagram showing the experimental design, timeline of mouse models and sampling strategy.\u003c/p\u003e\n\u003cp\u003e(D) Relative body weight of mice receiving DSS with antibiotics (DSS+Abx) and without (DSS) as shown in Fig B. n=5-6.\u003c/p\u003e\n\u003cp\u003e(E-G) Colonic morphologies (E), colon length (F) and serum H\u003csub\u003e2\u003c/sub\u003eS level (G) of mice under different treatments.\u003c/p\u003e\n\u003cp\u003e(H) Cystathionine beta-synthase (CBS) and cystathionine γ-lyase (CTH) protein levels were analysed by western blotting in mouse colon epithelial tissues. n=4.\u003c/p\u003e\n\u003cp\u003eSignificance was measured with two-way ANOVA with multiple comparisons.\u0026nbsp;\u0026nbsp; Nonparametric Mann-Whitney test was used for non-pairwise comparisons. Each dot represents an individual mouse.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/a96b2b598e10019cd76fa492.png"},{"id":53954704,"identity":"387eb64b-e6c3-47ad-9125-816c63b7f1ce","added_by":"auto","created_at":"2024-04-02 16:43:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":294444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGut bacterial ASR pathway contributes to H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS generation derived from dietary sulfated polysaccharide\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains formed H\u003csub\u003e2\u003c/sub\u003eS from λ-carrageenan. Molecular formula of λ-Carrageenan (above). Representatives of H\u003csub\u003e2\u003c/sub\u003eS detection using lead acetate strip after growing \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains in M9 medium supplemented with 1% λ-Carrageenan for 16h aerobically (below).\u003c/p\u003e\n\u003cp\u003e(B) Schematic outlining H\u003csub\u003e2\u003c/sub\u003eS production from DSS \u003cem\u003evia\u003c/em\u003e bacteria.\u003c/p\u003e\n\u003cp\u003e(C) DSS residue rate in mouse feces compared with abx-treated group in day3 and day5. Mice treatment was described in Fig. 5C. Unpaired t-test was used for non-pairwise comparisons.\u003c/p\u003e\n\u003cp\u003e(D) DSS degradation by fecal flora in mice and humans was tested in M9 supplemented with 1% DSS as the sole sulfur source. Medium without bacteria inoculation was used as controls.\u003c/p\u003e\n\u003cp\u003e(E) Flow chart of H\u003csub\u003e2\u003c/sub\u003eS production from DSS test (left). H\u003csub\u003e2\u003c/sub\u003eS production analysis by \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eP. mirabilis\u003c/em\u003e strains was examined using lead acetate strip (right).\u003c/p\u003e\n\u003cp\u003e(F) Growth of \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains on M9 supplemented with DSS as the sole sulfur source.\u003c/p\u003e\n\u003cp\u003e(G) DSS degradation by \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains was tested in M9 supplemented with 1% DSS as the sole sulfur source. Medium without bacteria inoculation was used as controls.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eValues are Mean ± SEM from at least three independent experiments. P-value was determined by ordinary one-way ANOVA analysis with multiple comparisons.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/b7a7acdc099ba1092482b2fb.png"},{"id":53954703,"identity":"0b52d67c-7a39-406d-9fb1-04f220502bbe","added_by":"auto","created_at":"2024-04-02 16:43:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":629356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ASR pathway modulates intestinal sulfation and DSS-induced colitis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp;Schematic diagram showing the experimental design, timeline of mouse models and sampling strategy.\u003c/p\u003e\n\u003cp\u003e(B,C) Serum H\u003csub\u003e2\u003c/sub\u003eS and caecum content SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2−\u003c/sup\u003e levels in mice colonized with \u003cem\u003eE. coli\u003c/em\u003e WT, \u003cem\u003e∆cysJ\u003c/em\u003e, and \u003cem\u003e∆cysM\u003c/em\u003e mutant strains. n = 3-5. Two cecum samples from the WT group were contaminated and excluded from the analysis.\u003c/p\u003e\n\u003cp\u003e(D) Body weight was tracked after DSS administration. n=5.\u003c/p\u003e\n\u003cp\u003e(E) Disease activity over the duration of the experiment.\u003c/p\u003e\n\u003cp\u003e(F) Colonic morphologies and representative H\u0026amp;E-Stained mouse colon sections at the termination of the experiment on day 6.\u003c/p\u003e\n\u003cp\u003e(G, H) Colon length (G) and histological assessment of disease severity (H).\u003c/p\u003e\n\u003cp\u003e(J) Representative micrographs of High-Iron Diamine-Alcian Blue (HID-AB) staining in mouse colon sections. Sulfomucin is stained black/brown and sialomucin is stained blue.\u003c/p\u003e\n\u003cp\u003e(K-L) Sulfomucin area ratio among \u003cem\u003e∆cysJ\u003c/em\u003e, \u003cem\u003e∆cysM\u003c/em\u003e and WT groups.\u003c/p\u003e\n\u003cp\u003e(L) Sulfomucin/Sialomucin ratio between \u003cem\u003e∆cysM\u003c/em\u003e and WT groups.\u003c/p\u003e\n\u003cp\u003e(M) The box-whiskers plot showed abundance of \u003cem\u003epapss2\u003c/em\u003e from CD and control subjects from FAH-SYSU, GSE-83687 and HMP cohorts. CPM, copy per million. The highest and lowest values are denoted by the upper and lower extremities of the vertical line, respectively, while the median is represented by the central horizontal line. * Refer to Fig. 2B for details regarding the control groups in each cohort.\u003c/p\u003e\n\u003cp\u003e(N) Relative mRNA levels of \u003cem\u003epapss2\u003c/em\u003e in the colonic tissue of the mice shown in Figure 7A. Data shown as mean ± SEM. Each point represents an individual mouse. Significance was measured with ordinary one- or two- way ANOVA analysis with multiple comparisons. Nonparametric Mann-Whitney test was used for non-pairwise comparisons.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/2459885ef89968150002f7c1.png"},{"id":63071293,"identity":"c598deb4-b1b6-4d6b-9fc4-bd3a738cf942","added_by":"auto","created_at":"2024-08-22 20:06:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4178958,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/a098e51f-39bd-455c-bc5a-805ad41133ec.pdf"},{"id":53954988,"identity":"5d8e86ba-fdf8-4acc-90d5-51e1ac4b5a8c","added_by":"auto","created_at":"2024-04-02 16:51:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1162651,"visible":true,"origin":"","legend":"","description":"","filename":"MicrobiomeSI032724.docx","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/ce868ad6828a054d9a40f6e9.docx"},{"id":53954696,"identity":"fe9357de-d86d-47d7-b553-412a901d80e9","added_by":"auto","created_at":"2024-04-02 16:43:13","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1092367,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydataset17.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4176488/v1/09c291bb1fc8f3b3cad6723a.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eMicrobial Assimilatory Sulfate Reduction-Mediated H\u003csub\u003e2\u003c/sub\u003eS: An Overlooked Role in Crohn's Disease Development\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCrohn's disease (CD) and ulcerative colitis (UC) are two main forms of Inflammatory Bowel Disease (IBD), characterized by symptoms including diarrhea, rectal bleeding, abdominal pain, fatigue, and weight loss, significantly impacting patients' lives. IBD incidence and prevalence are rising globally, particularly in newly industrialized regions\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The growing global burden of this disease underscores the need for preventive and therapeutic measures\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although the precise etiology remains elusive, it is believed to result from dysregulated mucosal immune responses triggered by gut bacteria, especially in individuals with genetic predispositions\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSulfur metabolism and sulfur-containing metabolites play a pivotal role in IBD \u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) is the sulfur derivative that garners the most attention in the context of colonic health. In the gastrointestinal system, the H\u003csub\u003e2\u003c/sub\u003eS pathway supports epithelial, immune, and enteric nervous system health through various mechanisms, including posttranslational modification of protein cysteine residues, activation of K\u003csub\u003eATP\u003c/sub\u003e channels, and serving as an inorganic fuel for colonocytes\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, excessive exposure to H\u003csub\u003e2\u003c/sub\u003eS can be detrimental to the host, damaging the intestinal epithelium and leading to chronic inflammation, as well as disrupting the balance between cellular proliferation and apoptosis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. An association between elevated H\u003csub\u003e2\u003c/sub\u003eS levels and IBD has long been suspected\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Several studies suggest that pharmacological interventions targeting H\u003csub\u003e2\u003c/sub\u003eS may improve outcomes in IBD through mechanisms such as driving regulatory T cell differentiation, stabilizing hypoxia-inducible factor 1-alpha (HIF-1α), promoting biofilm formation, and reducing planktonic bacteria growth\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, a comprehensive mechanistic model elucidating the relationship between H\u003csub\u003e2\u003c/sub\u003eS generation and IBD is still lacking. The production and release of H\u003csub\u003e2\u003c/sub\u003eS are regulated by both endogenous and exogenous factors, but the relative contributions of the host and gut microbiota to overall systemic H\u003csub\u003e2\u003c/sub\u003eS levels in humans remain uncertain. Endogenous H\u003csub\u003e2\u003c/sub\u003eS production primarily results from the enzymatic degradation of organic sulfur compounds, particularly cysteine. Key enzymes in this process include cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH), 3-mercaptopyruvate sulfur transferase (MPST), and methanethiol oxidase (SELENBP1). On the other hand, our understanding of microbial-mediated H\u003csub\u003e2\u003c/sub\u003eS generation remains limited\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBacteria produce H\u003csub\u003e2\u003c/sub\u003eS through the utilization of both organic sulfur compounds like L-cysteine and taurine, as well as inorganic sulfur compounds such as sulfate and sulfite. The two primary pathways for sulfate metabolism are Assimilatory Sulfate Reduction (ASR), involving the reduction of sulfate to H\u003csub\u003e2\u003c/sub\u003eS, which is subsequently incorporated into cysteine and methionine biosynthesis, and Dissimilatory Sulfate Reduction (DSR), a process found in sulfate-reducing bacteria where these microbes produce H\u003csub\u003e2\u003c/sub\u003eS from sulfate without integrating it into L-cysteine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious investigations into microbial sulfidogenesis found the contributions of gut microbiota to systemic total H\u003csub\u003e2\u003c/sub\u003eS levels varied widely across subjects\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and have mainly focused on the fermentation of organic sulfur compounds\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and sulfate-reducing bacteria DSR\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Meanwhile, ASR, a common strategy employed by many microbes to fix sulfur and manipulate organosulfur compounds, has been routinely overlooked.\u003c/p\u003e \u003cp\u003eHere, we employed genomic and metagenomic tools to gain a deeper understanding of the colonic sulfidogenic capacity of both the host and gut microbiome in a newly-onset treatment-na\u0026iuml;ve CD cohort, and observed that CD exhibit reduced endogenous H\u003csub\u003e2\u003c/sub\u003eS production alongside increased gut microbial H\u003csub\u003e2\u003c/sub\u003eS generation, primarily \u003cem\u003evia\u003c/em\u003e the ASR pathway. Mechanistically, we genetically manipulated \u003cem\u003eE. coli\u003c/em\u003e ASR pathway to evaluate the impact on (i) \u003cem\u003eE. coli\u003c/em\u003e's sulfidogenic capacity, (ii) colon epithelial cell viability, and (iii) the development of colitis and maintenance of mucus integrity in a mouse model. Our data elucidate a previously unappreciated role of microbial ASR pathway in dietary sulfate metabolism, intestinal sulfur homeostasis and mucus integrity, emphasizing its pivotal role in CD pathogenesis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCD is associated with up-regulation of gut microbial assimilatory sulfate reduction\u003c/h2\u003e\n \u003cp\u003eWe conducted a comprehensive investigation into the H\u003csub\u003e2\u003c/sub\u003eS production capabilities of the human gut microbiome, focusing on key genes responsible for sulfide generation from various sources, including organic compounds (such as dietary rich L-cysteine and taurine) and inorganic sulfate (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary dataset 1). Our analysis was based on stool metagenomic samples from two independent IBD cohorts, FAH-SYSU (treatment na\u0026iuml;ve IBD cohort enrolled at the First Affiliated Hospital of Sun Yat-sen University)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and PRISM (Prospective Registry of IBD study at MGH)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. ShortBRED was employed to identify unique sequence markers of related family members and quantifying their relative abundance in metagenomic data with high specificity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. It is noteworthy that ShortBRED was not specifically developed for gene cluster identification and quantification. Nevertheless, our search within metagenomic datasets using individual genes revealed a relatively high degree of consistency among genes from the same cluster (spearman r 0.59\u0026ndash;0.94, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Supplementary dataset 2), confirming the accuracy of the predictions.\u003c/p\u003e\n \u003cp\u003eWe found that genes associated with assimilatory sulfate reduction (ASR), including sulfate adenylyltransferase (CysND) and adenylylsulfate kinase (CysC), were prevalent (100% and 80\u0026ndash;100% in FAH-SYUS and PRISM cohort, respectively) and abundant (98.0-646.7 and 20.6\u0026ndash;57.2 RPKM in FAH-SYSU and PRISM cohort, respectively, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) in both CD patients and HC control subjects, although the PRISM cohort exhibited lower abundance, possibly due to differences in sequencing procedures. In the ASR pathway, organisms use different strategies: 1) Adenosine-5\u0026rsquo;-phosphosulfate (APS) is phosphorylated into 3\u0026rsquo;-Phosphoadenosine-5\u0026rsquo;-phosphosulfate (PAPS) by CysC, which is further reduced into sulfite (SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e) by PAPS reductase (CysH); 2) APS is directly reduced by an APS reductase (AprAB) to generate adenosine monophosphate (AMP) and SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e. Both scenarios generate SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e which could further be reduced by anaerobic sulfite reductase (AsrABC) or sulfite reductase (CysJI) to form sulfide (S\u003csup\u003e2\u0026minus;\u003c/sup\u003e), which subsequently yield L-cysteine mediated by cysteine synthase A (CysK\u003cem\u003e)\u003c/em\u003e and cysteine synthase B (CysM) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). We found ASR downstream genes, including \u003cem\u003ecysH\u003c/em\u003e, \u003cem\u003ecysJI\u003c/em\u003e, \u003cem\u003ecysM\u003c/em\u003e, \u003cem\u003ecysK\u003c/em\u003e, and \u003cem\u003easrABC\u003c/em\u003e, were also more abundant in CD subjects (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting a significant role for ASR in H\u003csub\u003e2\u003c/sub\u003eS production from SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e in CD individuals (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, Supplementary dataset 3).\u003c/p\u003e\n \u003cp\u003eWe observed that the prevalence and abundance of \u003cem\u003edsrAB\u003c/em\u003e genes (key genes for the DSR but not the ASR pathway) were notably lower compared to \u003cem\u003easr\u003c/em\u003e-associated genes in both cohorts. In the PRISM cohort, \u003cem\u003edsrAB\u003c/em\u003e genes were detected in approximately 30.9\u0026ndash;32.4% of CD subjects, while this percentage increased to 67.6\u0026ndash;76.5% in HC subjects. Moreover, their abundance increased from approximately 0.48 to 0.60\u0026ndash;0.72 RPKM (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, no significant difference in \u003cem\u003edsrAB\u003c/em\u003e genes was observed in the FAH-SYSU cohort (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, \u003cem\u003eaprAB\u003c/em\u003e, responsible for converting APS to SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e in both DSR and ASR, showed a marked reduction in CD subjects (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, we investigated \u003cem\u003ecysPUWA\u003c/em\u003e, which encodes a sulfate transporter common to both pathways. These transporter genes were more prevalent and abundant in CD subjects, indicating increased microbial sulfate transport in CD patients.\u003c/p\u003e\n \u003cp\u003eOrganic sulfur metabolism has been reported to be enriched in individuals with IBD and colorectal cancer (CRC) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Therefore, we investigated microbial genes associated with organic sulfur metabolism in our study. We found that the bacterial gene \u003cem\u003empst\u003c/em\u003e, crucial for converting L-cysteine to H\u003csub\u003e2\u003c/sub\u003eS, exhibited more prevalent and significantly elevated levels in CD subjects compared to HC subjects in both cohorts (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, genes related to taurine and alkanesulfonate metabolism, including taurine transporter (\u003cem\u003etauABC\u003c/em\u003e), taurine dioxygenase (\u003cem\u003etauD\u003c/em\u003e), sulfonate transporter (\u003cem\u003essuACB\u003c/em\u003e), and alkanesulfonate monooxygenase (\u003cem\u003essuD\u003c/em\u003e), were more abundant in CD subjects in FAH-SYSU cohort (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Microbial methanethiol oxidase (\u003cem\u003emtoX\u003c/em\u003e), widely distributed in the biosphere\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, was not detected in human associated bacteria, and therefore was excluded in the ShortBRED analysis. In the PRISM cohort, an increasing trend was observed in \u003cem\u003etauABC\u003c/em\u003e and \u003cem\u003essuD\u003c/em\u003e among CD subjects, although statistical significance was not attained.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eCD patients demonstrate impaired endogenous H\u003csub\u003e2\u003c/sub\u003eS production\u003c/h2\u003e\n \u003cp\u003eEndogenous H\u003csub\u003e2\u003c/sub\u003eS production arises from the host\u0026apos;s utilization of sulfur-containing amino acids (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). To shed light on endogenous sulfidogenic activity, we evaluated the expression levels of the host \u003cem\u003ecbs\u003c/em\u003e, \u003cem\u003ecth\u003c/em\u003e, \u003cem\u003empst\u003c/em\u003e and \u003cem\u003eselenbp1\u003c/em\u003e by examining intestinal biopsies obtained from newly diagnosed CD patients (n\u0026thinsp;=\u0026thinsp;46) and non-disease controls (n\u0026thinsp;=\u0026thinsp;44) from the FAH-SYSU cohort\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Our analysis revealed that all of these 4 genes exhibited significant decreases in inflamed mucosal biopsies from CD subjects (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). A similar trend was observed in three independent IBD cohorts, including the Mount Sinai Hospital cohort (GSE83687)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, a treatment-naive pediatric IBD cohort (E-MTAB-5464)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and the HMP IBD cohort\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), although statistical significance was not achieved in some cases. In the E-MTAB-5464 cohort, transcriptomic data were generated from purified intestinal epithelial cells. CBS raw counts in this cohort were generally less than 10, hence not analysed. This finding strongly suggests a substantial reduction in the endogenous sulfidogenic capacity of CD patients.\u003c/p\u003e\n \u003cp\u003eTo uncover whether CD patients have impaired H\u003csub\u003e2\u003c/sub\u003eS detoxification capacity, we examined the expression levels of key enzymes responsible for host H\u003csub\u003e2\u003c/sub\u003eS detoxification in these cohort datasets, including thiosulfate sulfurtransferase (TST), thiosulfate sulfide:quinone oxidoreductase (SQOR), and persulfide dioxygenase (ETHE1) (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our analysis revealed that \u003cem\u003etst\u003c/em\u003e was significantly downregulated, while \u003cem\u003esqor\u003c/em\u003e was upregulated in CD subjects in FAH-SYSU cohort. A similar trend was observed in the HPM IBD cohort, although \u003cem\u003etst\u003c/em\u003e did not reach statistical significance between CD and non-IBD groups in this cohort. The expression of \u003cem\u003eethe1\u003c/em\u003e remained similar in both FAH-SYSU and HMP cohorts, irrespective of CD or control groups (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Therefore, further research is needed to explore the H\u003csub\u003e2\u003c/sub\u003eS detoxification capacity in CD patients.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eOxygen-insensitive ASR is functionally more active in fecal microbiota from CD patients\u003c/h2\u003e\n \u003cp\u003eTo further substantiate the contribution of the ASR pathway from gut microbiota to H\u003csub\u003e2\u003c/sub\u003eS generation in CD, we conducted an \u003cem\u003eex vivo\u003c/em\u003e fecal culture experiment using thiosulfate (S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026thinsp;\u0026minus;\u0026thinsp;2\u0026minus;\u003c/sup\u003e) as the sole sulfur source. DSR has been reported in sulfate-reducing bacteria which are strictly anaerobes, whereas ASR has been reported in facultative anaerobes and aerobes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Therefore, we set up the fecal culture aerobically and measured H\u003csub\u003e2\u003c/sub\u003eS production in fecal samples from both healthy individuals and CD patients to test if oxygen-insensitive ASR activity was enhanced in CD\u0026rsquo;s gut microbiota. We detected H\u003csub\u003e2\u003c/sub\u003eS production in 19 out of 34 (55.9%) CD stool samples, with 13 showing notably high levels (\u0026gt;\u0026thinsp;10,000 intensity). In contrast, only 6 out of 35 (17.1%) samples from healthy controls exhibited H\u003csub\u003e2\u003c/sub\u003eS production, with 5 demonstrating high levels (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Thus, oxygen-insensitive ARS is more active in CD patients.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eThe bacterial ASR pathway is prevalent in the human microbiome\u003c/h2\u003e\n \u003cp\u003eThe \u003cem\u003easr\u003c/em\u003e gene cluster in \u003cem\u003eE. coli\u003c/em\u003e MG1655\u003csup\u003e33\u003c/sup\u003e and \u003cem\u003eSalmonella enterica\u003c/em\u003e ST8493\u003csup\u003e34\u003c/sup\u003e, along with the \u003cem\u003edsr\u003c/em\u003e gene cluster in \u003cem\u003eDesulfovibrio gigas\u003c/em\u003e DSM 1382\u003csup\u003e35\u003c/sup\u003e, that have been characterized in previous studies, are shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA. To comprehensively assess the distribution of \u003cem\u003easr\u003c/em\u003e- (\u003cem\u003ecysDN\u003c/em\u003e, \u003cem\u003ecysC\u003c/em\u003e, \u003cem\u003ecysH\u003c/em\u003e, \u003cem\u003ecysJI\u003c/em\u003e, \u003cem\u003ecysM, cysK\u003c/em\u003e, \u003cem\u003eaprAB and asrABC\u003c/em\u003e) and \u003cem\u003edsr\u003c/em\u003e-associated genes (\u003cem\u003edsrAB\u003c/em\u003e, \u003cem\u003eaprAB\u003c/em\u003e) among human bacteria, we screened these genes against 1635 Human Microbiome Project (HMP) reference genomes. This extensive analysis revealed that \u003cem\u003easr\u003c/em\u003e-associated genes are more widespread than \u003cem\u003edsrAB\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary dataset 4). A significant number (88.1%, 1441 out of 1635 reference genomes) of the total reference genomes contain at least one \u003cem\u003easr\u003c/em\u003e-associated gene, distributed predominantly in Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes, whilst \u003cem\u003edsrAB\u003c/em\u003e genes are only found in 0.43% (7 genomes) which are from Firmicutes and \u0026gamma;-Proteobacteria (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary dataset 4). The prevalence of \u003cem\u003ecysDN\u003c/em\u003e (409 genomes) and \u003cem\u003ecysC\u003c/em\u003e (295 genomes) in Bacteroidetes were higher than the other \u003cem\u003easr\u003c/em\u003e-associated genes. On the other hand, \u003cem\u003ecysJI\u003c/em\u003e (193 strains) and \u003cem\u003ecysM\u003c/em\u003e (275 strains) were more prevalent in Proteobacteria, including facultative aerobic species like \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eProteus. mirabilis\u003c/em\u003e, \u003cem\u003eKlebsiella. oxytoca\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary dataset 4). \u003cem\u003easrABC\u003c/em\u003e (117 genomes) was more commonly found in Firmicutes and Fusobacteria. These observations suggest a potential collaborative interplay among microorganisms in the execution of the ASR pathway.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eConstruction of\u003c/strong\u003e \u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003emutants with impaired assimilatory sulfate reduction (ASR)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWe focused on the \u003cem\u003ecysJI\u003c/em\u003e-mediated ASR pathway in this study since metagenomic data indicated that it is more abundant than \u003cem\u003easrABC\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). We used \u003cem\u003eE. coli\u003c/em\u003e MG1655, a known bacterium with a complete ASR pathway, as the model organism (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Using homologous recombination, we deleted two crucial ASR pathway genes, \u003cem\u003ecysJ\u003c/em\u003e and \u003cem\u003ecysM\u003c/em\u003e. \u003cem\u003ecysJ\u003c/em\u003e encodes sulfite reductase alpha subunit (\u003cem\u003ecysI\u003c/em\u003e encodes beta subunit), responsible for the reduction of SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026thinsp;\u0026minus;\u0026thinsp;2\u0026minus;\u003c/sup\u003e to S\u003csup\u003e2\u0026thinsp;\u0026minus;\u0026thinsp;2\u0026minus;\u003c/sup\u003e while \u003cem\u003ecysM\u003c/em\u003e encodes cysteine synthase B, which converts S\u003csup\u003e2\u0026minus;\u003c/sup\u003e to L-cysteine. (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). As expected, deleting \u003cem\u003ecysJ\u003c/em\u003e hindered \u003cem\u003eE. coli\u003c/em\u003e growth on SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e as the sole sulfur source (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eA). \u003cem\u003eE. coli\u003c/em\u003e carries a CysM homologue, CysK, which compensates for CysM in incorporating S\u003csup\u003e2\u0026minus;\u003c/sup\u003e into L-cysteine (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Consequently, \u003cem\u003eE. coli ∆cysM\u003c/em\u003e strains exhibited growth similar to the WT strain when SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e was the sole sulfur source. Alternatively, CysM can use thiosulfate (S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e) in place of S\u003csup\u003e2\u0026minus;\u003c/sup\u003e to produce L-cysteine \u003cem\u003evia\u003c/em\u003e S-sulfocysteine as the intermediate (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Therefore, \u003cem\u003eE. coli\u003c/em\u003e WT and \u003cem\u003e∆cysJ\u003c/em\u003e mutant grew on S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e as the sole sulfur source, however \u003cem\u003e∆cysM\u003c/em\u003e displayed diminished growth rate (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eWe assessed \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains for their sulfidogenic capabilities using various inorganic and organic sulfur sources. In the modified Sulfur, Indole, Motility (SIM) medium, we observed that deleting \u003cem\u003ecysJ\u003c/em\u003e increased H\u003csub\u003e2\u003c/sub\u003eS production from L-cysteine, while deleting \u003cem\u003ecysM\u003c/em\u003e enhanced H\u003csub\u003e2\u003c/sub\u003eS generation from both L-cysteine and SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). \u003cem\u003eE. coli\u003c/em\u003e WT also produced H\u003csub\u003e2\u003c/sub\u003eS from L-cysteine, as indicated by slight medium darkening (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). We further cultured \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains in M9 medium supplemented with varying concentrations of L-cysteine, and observed that all the strains exhibited a dose-dependent production of H\u003csub\u003e2\u003c/sub\u003eS, with the \u003cem\u003e∆cysJ\u003c/em\u003e mutant demonstrating greater efficiency in converting L-cysteine to sulfide than \u003cem\u003e∆cysM\u003c/em\u003e and WT (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). The enhanced H\u003csub\u003e2\u003c/sub\u003eS productivity of the \u003cem\u003e∆cysJ\u003c/em\u003e mutant remained consistent under anaerobic condition (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eC). Additionally, the \u003cem\u003e∆cysJ\u003c/em\u003e mutant accumulated SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e in M9 medium when SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e or S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e was the sole sulfur source due to the loss of sulfite reductase activity (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). These findings highlight the impact of ASR pathway alterations on both inorganic and organic sulfur metabolism. \u003cem\u003eE. coli\u003c/em\u003e WT and ASR-deficient mutants displayed distinct morphological characteristics and proteomic profiles (Fig. S3A, B, Supplementary dataset 5), suggesting that the alteration of the ASR pathway has a profound effect on bacterial physiology.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eBacterial assimilatory sulfate reduction modulates epithelial cell viability\u003c/h2\u003e\n \u003cp\u003eWe proceeded to investigate the impact of modifications in the bacterial ASR pathway on the growth of colonic epithelial cells in an \u003cem\u003ein vitro\u003c/em\u003e setting. We co-cultured \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains with normal human colonic mucosal epithelial cell line NCM460 with either L-cysteine or S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e as the sole sulfur source. Cell viability assay revealed that in the presence of L-cysteine, the \u003cem\u003e∆cysJ\u003c/em\u003e mutant led to significantly decreased cell viability, concurrent with increased H\u003csub\u003e2\u003c/sub\u003eS production (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, F). When S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e served as the exclusive sulfur source, the \u003cem\u003e∆cysM\u003c/em\u003e mutant induced more pronounced cell death, accompanied by higher H\u003csub\u003e2\u003c/sub\u003eS generation (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, F).\u003c/p\u003e\n \u003cp\u003eAs \u003cem\u003e∆cysJ\u003c/em\u003e mutant accumulates more SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e in the medium when SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e is the sole sulfur source (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE), this accumulation of SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e may potentially leads to cell toxicity \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. To investigate it further, we collected the supernatants from cultures of \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains grown in M9 medium supplemented with Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and used them to treat NCM460 cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). As indicated by the cell proliferation assay, the \u003cem\u003e∆cysJ\u003c/em\u003e mutant exhibited the most pronounced inhibition of cell proliferation in agreement with the high levels of SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH). Thus, the data suggest that bacterial ASR modulates epithelial cell viability through SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e metabolites.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eThe gut microbiota is the primary contributor to serum H\u003c/strong\u003e \u003csub\u003e\u0026nbsp;\u003cstrong\u003e2\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003eS levels in the DSS-induced mouse colitis model\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eColitis, a key component of IBD, is frequently studied using murine models. One widely employed method to induce colitis in these models is the administration of dextran sodium sulfate (DSS) \u003cem\u003evia\u003c/em\u003e drinking water. Our initial objective was to determine if H\u003csub\u003e2\u003c/sub\u003eS production is linked to the DSS-induced colitis model. We found a significant increase in serum H\u003csub\u003e2\u003c/sub\u003eS levels in mice received DSS compared to vehicle controls (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), suggesting that serum H\u003csub\u003e2\u003c/sub\u003eS is associated with DSS-induced colitis.\u003c/p\u003e\n \u003cp\u003eAlthough a previous study suggested that germ-free mice exhibit reduced plasma H\u003csub\u003e2\u003c/sub\u003eS levels\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, the specific contribution of the gut microbiota to systemic H\u003csub\u003e2\u003c/sub\u003eS levels in the context of DSS-induced colitis remained unknown. To illuminate the link between elevated serum H\u003csub\u003e2\u003c/sub\u003eS and gut microbes, we performed two studies. First, we utilized a publicly available colonic tissue transcriptomic dataset from mice undergoing DSS-induced colitis, followed by a tissue regeneration phase (GSE131032)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. During the colitis and recovery stages, the expression of \u003cem\u003ecbs\u003c/em\u003e and \u003cem\u003ecth\u003c/em\u003e genes remained stable, while \u003cem\u003empst\u003c/em\u003e and \u003cem\u003eselenbp1\u003c/em\u003e expression displayed a decreasing trend during colitis, followed by a slight elevation during the recovery stage (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). This suggested that endogenous H\u003csub\u003e2\u003c/sub\u003eS production remained consistent or even decreased during DSS-induced colitis, hence the rise in serum H\u003csub\u003e2\u003c/sub\u003eS observed is probably from gut microbiota. Second, we administered broad spectrum antibiotics (Abx) to mice in the DSS-induced model (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), and observed a significant reduction in serum H\u003csub\u003e2\u003c/sub\u003eS levels and alleviated DSS-induced colitis, as evidenced by weight and colon length measurements (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD-G). Given mRNA levels of \u003cem\u003ecbs\u003c/em\u003e and \u003cem\u003ecth\u003c/em\u003e remained stable throughout the DSS-induced colitis and recovery stages (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), we further examined protein levels of CBS and CTH in Abx-challenge mice experiment, and observed no significant difference between the two groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). Collectively, these findings provide compelling evidence that the gut microbiota plays a central role in the elevation of systemic H\u003csub\u003e2\u003c/sub\u003eS levels in the DSS-induced colitis model. Therefore, we utilized this model to investigate the causal relationship between microbial ASR pathway and colitis \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eThe gut bacterial ASR pathway contributes to sulfide generation derived from dietary sulfate\u003c/h2\u003e\n \u003cp\u003eDiet plays a pivotal role in shaping the composition and metabolic activity of the gut microbiota. While prior research mainly concentrated on organic sulfur compounds from dietary proteins, the role of inorganic sulfur (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e) remains understudied\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Carrageenan, a common sulfated polysaccharide food additive, is linked to UC relapse risk and can induce intestinal inflammation in animal model\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. We hypothesized that gut microbiota-mediated carrageenan degradation and subsequent H\u003csub\u003e2\u003c/sub\u003eS production might contribute to its pro-colitis effects.\u003c/p\u003e\n \u003cp\u003eTo test this hypothesis, we initially cultured \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains in M9 medium supplemented with \u0026lambda;-carrageenan as the sole sulfur source, owing to its high sulfur content (32\u0026ndash;39%, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Surprisingly, both WT and mutant strains demonstrated H\u003csub\u003e2\u003c/sub\u003eS production (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA), which contrasted with previous findings that \u003cem\u003e∆cysJ\u003c/em\u003e mutant couldn\u0026rsquo;t grow on inorganic SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e. Given that carrageenan is a biopolymer derived from red algae, it likely contains trace amounts of organic sulfur compounds that can be utilized by \u003cem\u003e∆cysJ\u003c/em\u003e mutant. As a result, we transitioned to DSS, a synthetic sulfated polysaccharide with approximately 18\u0026ndash;20% sulfur content (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB), which as mentioned earlier, is a commonly used as inducer in murine colitis models\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eWe first tested whether the gut microbiota was involved in DSS degradation. Abx-treated mice exhibited significantly higher fecal DSS levels compared to vehicle control mice, suggesting active DSS degradation by gut microbiota \u003cem\u003ein vivo\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). \u003cem\u003eEx vivo\u003c/em\u003e experiments with mouse and human stool samples showed about 35% of the DSS was consumed after overnight incubation (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD), confirmed microbiota-mediated DSS degradation.\u003c/p\u003e\n \u003cp\u003eWe postulated that DSS degradation releases SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, which are subsequently assimilated by bacteria employing the ASR pathway. To test this hypothesis, we initiated an experiment involving 1% DSS incubation with human/mouse fecal cultures for 16 hours, followed by supernatant collection, and subsequent inoculation with \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). As anticipated, \u003cem\u003eE. coli\u003c/em\u003e WT and mutant strains formed H\u003csub\u003e2\u003c/sub\u003eS in the presence of pre-incubated DSS. \u003cem\u003eE. coli ∆cysM\u003c/em\u003e mutant produced higher levels of H\u003csub\u003e2\u003c/sub\u003eS than WT and the \u003cem\u003e∆cysJ\u003c/em\u003e mutant (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). \u003cem\u003eProteus mirabilis\u003c/em\u003e, which carries the \u003cem\u003easr\u003c/em\u003e-gene cluster, generated H\u003csub\u003e2\u003c/sub\u003eS as well (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE, Supplementary dataset 4). Direct culture of \u003cem\u003eE. coli ∆cysJ\u003c/em\u003e mutant in M9 medium with DSS as the sole sulfur source did not yield growth (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). Although \u003cem\u003eE. coli\u003c/em\u003e WT and \u003cem\u003e∆cysM\u003c/em\u003e mutant grew on DSS as the sole sulfur source, they showed low utilization and negligible DSS degradation, emphasizing metabolic cross-feeding among bacterial species for efficient sulfated polysaccharide metabolism (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF-G).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eThe ASR pathway modulates DSS-induced colitis\u003c/strong\u003e \u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eSPF mice received Abx-cocktail were subsequently inoculated with \u003cem\u003eE. coli\u003c/em\u003e WT, \u003cem\u003e∆cysJ\u003c/em\u003e and \u003cem\u003e∆cysM\u003c/em\u003e, then subjected to DSS administration (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Mice colonized with the \u003cem\u003eE. coli ∆cysJ\u003c/em\u003e mutant exhibited elevated serum H\u003csub\u003e2\u003c/sub\u003eS and fecal SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e levels associated with more severe disease phenotype evidenced by a greater body weight loss, a worsening of disease activity, and more severe intestinal inflammation characterized by increased mucosal erosion, crypt destruction and inflammatory cell infiltration in the colon (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB-H). The heightened serum H\u003csub\u003e2\u003c/sub\u003eS levels may be ascribed to the degradation of organic sulfur compounds within the gastrointestinal tract, such as L-cysteine, catalyzed by \u003cem\u003eE. coli ∆cysJ\u003c/em\u003e. We found a significant reduction in plasma levels of ursodeoxycholic acid (UDCA), \u0026alpha;- and \u0026omega;-muricholic acid (MCA), and an increase in cholic acid-7-sulfate (CA-7S) in mice colonized with the \u003cem\u003e∆cysJ\u003c/em\u003e strain (Fig. S4A), indicating that alteration of \u003cem\u003eE. coli\u003c/em\u003e ASR pathway strongly influenced the bile acid profile in mice.\u003c/p\u003e\n \u003cp\u003eWhile \u003cem\u003e∆cysM\u003c/em\u003e and WT-colonized mice exhibited similar disease severity, serum H\u003csub\u003e2\u003c/sub\u003eS and fecal SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e levels, a significant difference in colonic mucin composition was observed. The colonic mucus layer, essential for maintaining homeostasis between resident microbiota and underlying immune cells, is primarily composed of acidomucins, broadly categorized as sialomucins or sulfomucins depending on the presence of sialic acid or sulfate groups\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Colonic tissues from \u003cem\u003e∆cysM\u003c/em\u003e-colonized mice showed a reduced sulfomucin:sialomucin ratio in (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eI-K), indicating compromised host sulfation than WT mice. Intestinal sulfation, crucial for colitis protection, is dependent on the host PAPS synthase 2 (PAPSS2), which is central in generating PAPS, the universal sulfonate donor for sulfation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Our analysis of transcriptomic data from the FAH-SYSU, HMP and GSE83687 cohorts indicated a significant decrease in colonic \u003cem\u003epapss2\u003c/em\u003e gene expression in actively inflamed CD patients compared to non-disease and non-IBD controls (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eL). Mice deficient in \u003cem\u003epapss2\u003c/em\u003e have been previously demonstrated to manifest reduced intestinal sulfomucin content, rendering them susceptible to DSS-induced colitis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Therefore, we reasoned that reduced host sulfate in \u003cem\u003e∆cysM\u003c/em\u003e-colonized mice were due to the downregulation of \u003cem\u003epapss2\u003c/em\u003e. Indeed, we observed decreased colonic mRNA expression of \u003cem\u003epapss2\u003c/em\u003e in both \u003cem\u003e∆cysJ\u003c/em\u003e and \u003cem\u003e∆cysM\u003c/em\u003e groups compared to the WT group, as confirmed by real-time PCR analysis (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eM).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe burden of IBD, which encompasses conditions like CD and UC, is substantial and often leads to hospitalizations and surgical interventions\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Current treatments primarily target host inflammatory pathways using non-specific immunosuppressive agents, which can pose significant risks and may not always be effective, necessitating the exploration of alternative approaches\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Mounting evidence suggests that an imbalance in H\u003csub\u003e2\u003c/sub\u003eS production, either insufficient or excessive, can act as an environmental trigger for CD \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Studies have shown that the administration of H\u003csub\u003e2\u003c/sub\u003eS donors can suppress the expression of proinflammatory cytokines and ameliorate colitis in murine models\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This raises the possibility that modulating H\u003csub\u003e2\u003c/sub\u003eS concentrations in the gut lumen could be an exciting therapeutic strategy for treating CD\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, research into this potential link between H\u003csub\u003e2\u003c/sub\u003eS and CD has been hindered by a limited understanding of sulfur metabolism within the human gut.\u003c/p\u003e \u003cp\u003eTo address this knowledge gap, we conducted a comprehensive investigation into the functional capacity of both the gut microbiota and host in H\u003csub\u003e2\u003c/sub\u003eS production. Our findings suggest that microbial sulfur metabolism within the human colon is more complex and widespread than previously recognized. We analyzed metagenomic data from independent IBD cohorts and found that CD is associated with an increase in microbial generation through ASR pathways, as evidenced by the increased abundance and prevalence of \u003cem\u003easr\u003c/em\u003e-associated genes. \u003cem\u003eEx vivo\u003c/em\u003e fecal culture confirmed ASR-mediated H\u003csub\u003e2\u003c/sub\u003eS generation is more functionally active in stool samples from CD patients. Using \u003cem\u003eE. coli\u003c/em\u003e as the model organism, we generated \u003cem\u003e∆cysJ\u003c/em\u003e and \u003cem\u003e∆cysM\u003c/em\u003e mutants deficient in the ASR pathway. We conducted \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies to validate that the bacterial ASR pathway modulates cell viability, host sulfate homeostasis, and colitis pathogenesis. Our investigation has brought into focus the pivotal role played by ASR pathway in reshaping the utilization of L-cysteine and generation of H\u003csub\u003e2\u003c/sub\u003eS. The deletion of \u003cem\u003ecysJ\u003c/em\u003e gene in \u003cem\u003eE. coli\u003c/em\u003e amplifies H\u003csub\u003e2\u003c/sub\u003eS production from L-cysteine. The heightened metabolism of L-cysteine by gut microbes and increased abundance of \u003cem\u003ecysM\u003c/em\u003e has recently been associated with CRC\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We noticed that \u003cem\u003easrABC\u003c/em\u003e is enriched in CD subjects, warranting further investigation into its potential association with CD.\u003c/p\u003e \u003cp\u003eIn contrast to previous research, our findings suggest that the DSR pathway is unlikely to be the primary contributor to the elevated fecal microbial sulfidogenic capacity in CD. Prior research on exogenous H\u003csub\u003e2\u003c/sub\u003eS generation primarily centered on DSR, based on the culturing and sequencing of \u003cem\u003eDesulfovibrio\u003c/em\u003e genus, sulfate-reducing bacteria frequently found in the human and animal gut\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. However, Anantharaman et al.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e revealed that \u003cem\u003edsrAB\u003c/em\u003e-mediated dissimilatory sulfur metabolism is predicted in a much broader diversity of bacterial and archaeal groups than previously recognized, primarily due to horizontal gene transfer, such as \u003cem\u003eBilophila wadsworthia\u003c/em\u003e, an opportunistic pathogen inhabiting the gut. Consequently, it is more reasonable to predict DSR-mediated H\u003csub\u003e2\u003c/sub\u003eS generation based on \u003cem\u003edsr\u003c/em\u003e-gene cluster quantification, rather than relying solely on \u003cem\u003eDesulfovibrio\u003c/em\u003e quantification.\u003c/p\u003e \u003cp\u003eAnalysis of intestinal biopsy transcriptomic data from multiple IBD cohorts has unveiled a compromised endogenous sulfidogenic capacity in CD patients. This is evident from the downregulation of key genes, specifically \u003cem\u003ecbs\u003c/em\u003e, \u003cem\u003ecth\u003c/em\u003e, \u003cem\u003empst\u003c/em\u003e and \u003cem\u003eselenbp1\u003c/em\u003e. Severe CD manifestations in a child with \u003cem\u003ecbs\u003c/em\u003e deficiency has been reported\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Reduced expression of \u003cem\u003ecbs mpst\u003c/em\u003e and \u003cem\u003eselenbp1\u003c/em\u003e has been linked to the exacerbation of inflammation-induced intestinal barrier injury in UC and CD\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003c/sup\u003e. Animal studies have provided additional evidence highlighting the critical role of endogenous H\u003csub\u003e2\u003c/sub\u003eS generation in colitis. MPST\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and MPST\u003csup\u003e+/\u0026minus;\u003c/sup\u003e mice exhibit exacerbated DSS-induced colitis\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Inhibition of endogenous H\u003csub\u003e2\u003c/sub\u003eS synthesis through the use of CBS and CTH inhibitors, such as β-cyanoalanine, propargylglycine, and O-carboxymethyl-hydroxylamine hemihydrochloride, has been demonstrated to worsen colitis in mouse model\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Further investigations are warranted to elucidate the factors responsible for the downregulation of genes involved in endogenous H\u003csub\u003e2\u003c/sub\u003eS generation.\u003c/p\u003e \u003cp\u003eConsiderable efforts are underway to investigate the potential contributions of dietary factors to the pathophysiology of IBD\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The interplay of genetic, environmental, microbial, and immunological factors makes diet a crucial aspect of IBD etiology\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Dietary sulfur intake, primarily from inorganic sulfate and sulfur-containing amino acids (SAAs) such as methionine, cysteine, and taurine, plays a significant role\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, estimations of dietary sulfur content often fail to account for sulfur-containing food modifiers or additives, such as carrageenan and sulfiting agents (\u003cem\u003ee.g.\u003c/em\u003e, potassium bisulfate, sodium bisulfate)\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Daily intake of inorganic sulfate is estimated to range from 1.5 to 16.0 mmol \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Interestingly, fecal sulfate (and sulfide) excretion is minimal compared to dietary intake, suggesting that sulfate is actively removed from the fecal stream during passage through the gut by both host and gut microbiota\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Carrageenan, a sulfated polysaccharide, contains approximately 15\u0026ndash;40% sulfur, depending on the specific type of carrageenan (\u003cem\u003ee.g.\u003c/em\u003e, kappa, iota, lambda) and the seaweed species used for extraction\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Carrageenan is widely used as a food additive in the Western diet, and its consumption has substantially increased over the past 50 years, paralleling the rising prevalence of IBD\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Successful dietary interventions that induced CD remission have excluded processed foods containing carrageenan, further supporting the notion that carrageenan may trigger or exacerbate inflammation in IBD\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In animal models, carrageenan administration consistently induces intestinal ulcerations resembling human IBD histopathologically\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. We reasoned that SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e released during carrageenan degradation are utilized by gut microbes, contributing to IBD pathogenesis. To investigate, we used synthetic sulfated polysaccharide DSS due to carrageenan's organic sulfur contaminants. The DSS-induced colitis model is known for consistently mimicking epithelial damage seen in IBD, underscoring the complex interplay between dietary elements, gut microbiota, and disease pathogenesis\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. DSS mouse colitis model is known for its variability even among genetically identical mice and across different mouse facilities. A recent study found that gut microbiota plays a significant role in driving this variability within the model\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThrough \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e studies, we've demonstrated that the gut microbiota can degrade DSS, releasing SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e that fuel bacterial ASR pathways. This alteration in microbial sulfur metabolism ultimately modulates disease severity. Our study highlights the crucial role of gut microbial ASR metabolism in dietary sulfate metabolism and susceptibility to colitis. It's important to acknowledge the significant daily intake of inorganic sulfate and the potential exacerbation of microbial H\u003csub\u003e2\u003c/sub\u003eS production by carrageenan used as food additives in processed foods, which can lead to mucosal damage. Furthermore, it is worth noting that CD pathogenesis is unlikely to be solely attributed to the presence and activities of single species. Genes associated with the ASR pathway exhibited varying distribution patterns among human bacteria. Additionally, in DSS degradation, mouse and human fecal microbial communities are more efficient than \u003cem\u003eE. coli\u003c/em\u003e monocultures, suggesting cross-feeding among different bacterial species for the efficient metabolism of sulfated polysaccharides.\u003c/p\u003e \u003cp\u003eIn summary, our study reveals the extensive diversity of microbial sulfur metabolism pathways. These findings highlight the association between CD and reduced endogenous H\u003csub\u003e2\u003c/sub\u003eS production alongside increased gut microbial H\u003csub\u003e2\u003c/sub\u003eS generation, primarily \u003cem\u003evia\u003c/em\u003e the ASR pathway. Microbial ASR-mediated dietary sulfate metabolism emerges as a crucial factor in colitis. Our research sheds light on the complex interaction between diet, the gut microbiota, and inorganic sulfate metabolism, highlighting their potential as promising therapeutic targets for managing CD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHuman subjects\u003c/h2\u003e \u003cp\u003e All study protocols abided by the Declaration of Helsinki principles and were approved by Ethical Committees of the First Affiliated Hospital of Sun Yat-sen University. Intestinal biopsies and stool specimens were collected as part of the FAH-SYSU cohort study (2016[113]). Subject stool samples were collected at the FAH, SYSU gastroenterology clinic and stored at -80\u0026deg;C immediately. For culturing assays, fecal samples were collected and diluted to make a 10% (w/v) fecal slurry by resuspension of the feces in 10% (w/v) glycerol solution, and aliquots were stored in cryogenic vials at -80\u0026deg;C until use. The exclusion criteria applied to all groups were as follows: recent (\u0026lt;\u0026thinsp;3 months prior) use of any antibiotic therapy, current extreme diet (\u003cem\u003ee.g.\u003c/em\u003e, parenteral nutrition or macrobiotic diet), known history of malignancy, current consumption of probiotics, any gastrointestinal tract surgery leaving permanent residua (\u003cem\u003ee.g.\u003c/em\u003e, gastrectomy, bariatric surgery, colectomy), or significant liver, renal, or peptic ulcer disease.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalyses of\u003c/b\u003e \u003cb\u003easr\u003c/b\u003e\u003cb\u003e- and\u003c/b\u003e \u003cb\u003edsr\u003c/b\u003e\u003cb\u003e-associated genes in Human Microbiome project (HMP) references genomes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHMP references genomes (1635 genomes as of June 30, 2023) were selected and analyzed through the IMG program on the Joint Genome Institute website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://img.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://img.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e63\u003c/sup\u003e. The functions (Supplementary dataset 1) were used to carry out a \u0026ldquo;Function Profile\u0026rdquo; against all selected reference genomes to identify those carrying \u003cem\u003easr\u003c/em\u003e- and \u003cem\u003edsr\u003c/em\u003e-associated genes. Hits were manually inspected. Genomes carrying sulfidogenic gene(s) were selected to generate a phylogenetic tree using phyloT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phylot.biobyte.de/\u003c/span\u003e\u003cspan address=\"https://phylot.biobyte.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on NCBI taxonomy and visualized using iTOL\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Genome and gene IMG ID are available in Supplementary dataset 4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMetagenomic data analysis\u003c/h2\u003e \u003cp\u003eWe used ShortBRED\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e to accurately profile the abundance of genes involved in the H\u003csub\u003e2\u003c/sub\u003eS generation in metagenomes sourced from the FAH-SYSU (BioProject: PRJNA793776)\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e and PRISM (BioProject: PRJNA400072)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e datasets. We initially compiled a set of identified bacterial sulfidogenic genes as our query sequences (Supplementary dataset 6). Subsequently, ShortBRED-Identify was employed to generate markers for these key bacterial sulfidogenic gene sequences using UniRef90 (May, 2023) as a reference list with an 85% cluster ID threshold. These markers were applied in ShortBRED-Quantify to assess gene abundance in paired metagenomes, which had previously undergone quality control \u003cem\u003evia\u003c/em\u003e the KneadData workflow (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://huttenhower.sph.harvard.edu/kneaddata\u003c/span\u003e\u003cspan address=\"http://huttenhower.sph.harvard.edu/kneaddata\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The output from ShortBRED-Quantify was expressed as reads per million reads per kilobase million (RPKM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCultivation of wild type bacteria and mutants\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e MG1655 wild type, mutants (Δ\u003cem\u003ecysJ and\u003c/em\u003e Δ\u003cem\u003ecysM\u003c/em\u003e) and Proteus mirabilis ATCC 29906 were generally cultivated in Luria broth (LB) containing tryptone (10 g\u0026middot;l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), yeast extract (5 g\u0026middot;l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and NaCl (10 g\u0026middot;l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). To characterize the growth of \u003cem\u003eE. coli\u003c/em\u003e wild type and mutant strains, they were cultivated in 5 mL LB overnight at 37\u0026deg;C in a shaking incubator (250 rpm) and the pellet was collected by centrifugation at 3,000\u0026times;g for 10 min. Cell pellets were then washed and re-suspended in fresh M9 media (inoculum size 1:20, v/v). The defined M9 medium contained NaCl (0.5 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (3 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO (6 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), NH\u003csub\u003e4\u003c/sub\u003eCl (1 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), MgCl\u003csub\u003e2\u003c/sub\u003e (95 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CaCl\u003csub\u003e2\u003c/sub\u003e (11.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and glucose (0.1%, w/v). 1 mM Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, L-cysteine or DSS was used as sole sulfur source. 200 \u0026micro;L samples were collected from each tube at the indicated time points, and their optical density at 600 nm was measured in flat-bottom 96-well plates (200 \u0026micro;L per well). Sulfite in the supernatant was quantified as described in \u003cspan refid=\"Sec16\" class=\"InternalRef\"\u003eSulfite Quantification\u003c/span\u003e section.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAllele-exchange mutagenesis of\u003c/b\u003e \u003cb\u003eΔcysJ\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eΔcysM\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eMG1655\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDNA fragments (~\u0026thinsp;1 kb) corresponding to the upstream and downstream regions of the target gene were amplified and a subsequent overlap PCR was used to fuse the two fragments which were then ligated into suicide plasmid harboring kanamycin resistance cassette, oriT (mob), \u003cem\u003esacB\u003c/em\u003e counter selection marker and R6K origin of replication using the In-Fusion HD Cloning kit (Clontech). The ligated suicidal plasmid (pKmobSac) was transformed into the donor strain, \u003cem\u003eE. coli\u003c/em\u003e S17 λpir. In parallel, \u003cem\u003eE. coli\u003c/em\u003e MG1655 was transformed with a temperature-sensitive ampicillin-resistance plasmid carrying oriR101 origin of replication (p101-Amp). The suicidal plasmid (pKmobSac) was then transformed into \u003cem\u003eE. coli\u003c/em\u003e MG1655 through conjugation and the resulted conjugants were screened at room temperature on LB agar plates containing ampicillin at 100 ng/\u0026micro;L (to select against \u003cem\u003eE. coli\u003c/em\u003e S17 donor cells) and kanamycin at 50 ng/\u0026micro;L. One single-crossover integrant was then selected and re-streaked on LSW-Sucrose agar plate (tryptone 10 g/L, yeast extract 5 g/L, glycerol 5 mL/L, NaCl 0.4 g/L, sucrose 100 g/L and agar 20 g/L)\u003csup\u003e66\u003c/sup\u003e supplemented with ampicillin at 100 ng/\u0026micro;L to select for the correct double cross-over mutants. One mutant was then selected, re-streaked, and confirmed for the loss of the conjugated plasmid through Sanger sequencing and its ability to grow in presence of ampicillin but not kanamycin. The knockout \u003cem\u003eE. coli\u003c/em\u003e MG1655 mutants were then cured from p101-amp plasmid through growing at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eH\u003csub\u003e2\u003c/sub\u003eS quantification\u003c/h2\u003e \u003cp\u003ePlasma H\u003csub\u003e2\u003c/sub\u003eS levels were quantified using a modified methylene blue method. In brief, ZnAC was added to 100 \u0026micro;L of plasma samples to precipitate H\u003csub\u003e2\u003c/sub\u003eS, HS\u003csup\u003e\u0026minus;\u003c/sup\u003e, S\u0026sup2;-, and plasma proteins. Subsequently, the ZnS pellet was re-dissolved by adding 130 \u0026micro;L of 2% N, N-dimethyl-p-phenylenediamine and 130 \u0026micro;L of 20% trichloroacetic acid. Methylene blue formation was initiated by addition of FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO and quantified at 665 nm using a spectrophotometer. Microbial culture H\u003csub\u003e2\u003c/sub\u003eS levels were quantified by Modified \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS\u003c/span\u003eulfur, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eI\u003c/span\u003endole, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eM\u003c/span\u003eotility (SIM)-medium and Lead Acetate Test Strip. See online supplemental material for further details.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDextran sulfate quantification\u003c/h2\u003e \u003cp\u003eTo measure DSS concentration in mouse fecal pellets, Sample aliquots (50 \u0026micro;L) were injected onto a size exclusion column (SEC-150, 3 \u0026micro;M, 7.8 \u0026times; 300 mm, Welch, Cat # 00237-21052) and eluted at a flow rate of 1.5 mL/min. The mobile phase consisted of 25 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 25 mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO, 50 mM KCl, and 10% ethanol. The eluent passed through a post-column derivatization instrument (LABRAT, LYM-1060), where it mixed with a 10 \u0026micro;g/mL dimethylene blue zinc chloride double salt (DMB, Sigma, 34108) delivered directly by pump A connected to the online mixer. Detection was performed using a VWD detector at 530 nm wavelength, with data collected \u003cem\u003evia\u003c/em\u003e OpenLAB CDS chromatography data software (Agilent, 1260 Infinity II).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSulfite quantification\u003c/h2\u003e \u003cp\u003e150 \u0026micro;L of culture supernatant or caecum slurry extract was mixed with 350 \u0026micro;L distilled water and 10 \u0026micro;L 10 M NaOH. Sulfite was quantified using the Total Sulfite Assay Kit (JC-HX-04, HK) based on the Pararosaniline Method, following the manufacturer's instructions. The reaction formed a purple-red complex, pararosaniline methylsulfonic acid, which exhibited maximal absorption at 550 nm, and absorbance was measured after a 10-minute incubation using a plate reader (UV-2450, SHIMADZU, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and viability assay\u003c/h2\u003e \u003cp\u003eThe NCM460 human colon epithelial cell line (RRID: CVCL_0460) was maintained in RPMI 1640 basic medium (gibco, 8123133) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 100 \u0026micro;g/mL penicillin G, and 100 \u0026micro;g/mL streptomycin sulfate (Invitrogen, Carlsbad, CA, USA). Cells were cultured at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator.\u003c/p\u003e \u003cp\u003eCell viability was analyzed by live/dead staining or Cell Counting Kit-8 (CCK8, GLPBIO, GK10001) according to the manufacturer's instructions. For live/dead staining, NCM460 cells (4 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e/well) in 96-well microplates were cultured for 48 hours in 100 \u0026micro;L of 1640 medium. After washing with PBS, cells were prepared for bacterial co-culture. \u003cem\u003eE. coli\u003c/em\u003e strains were cultivated overnight at 37\u0026deg;C in LB, washed in sulfur-free M9 media, and resuspended in M9 medium with L-cysteine or Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e as the sole sulfur source. Cells were co-cultured with these bacteria for 2 hours (150 \u0026micro;L bacterial culture per well). Afterward, cells were stained with 30 \u0026micro;L calcein-AM/PI working solution (2 \u0026micro;M calcein-AM and 4.5 \u0026micro;M propidium iodide) at 37\u0026deg;C for 20 minutes, followed by fixation with 0.4% polyformaldehyde. Quadruplicate experiments were conducted, and images of cells were acquired and analyzed using an Olympus IX83 fluorescence microscope. All experiments were performed in quadruplicate. The images of the cells were acquired immediately and analyzed by using a fluorescence microscope (Olympus IX83). Positive cell percentages and average fluorescence intensity were determined using Image-Pro Plus 6.0. For CCK8 assay, NCM460 cells (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e/well) were cultured overnight in 96-well microplates and treated with 50 \u0026micro;L of bacterial supernatants from M9 medium cultures with 2 mM Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as the sole sulfur source. After a 6-hour incubation, cells were washed, incubated with 100 \u0026micro;L of RPMI 1640 Medium plus 10 \u0026micro;L CCK-8 reagent, and absorbance at 450 nm was monitored. All experiments were performed in six replicates and blank wells without cells served as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Studies\u003c/h2\u003e \u003cp\u003eMale SPF C57BL/6 mice (6\u0026ndash;8 weeks) were maintained on a standard normal rodent diet (Synergy Bio, AIN-93M). All the mice used in this study were bred and raised in the animal facility of the First Affiliated Hospital of Sun Yat-sen University. Mice (n\u0026thinsp;=\u0026thinsp;6) received antibiotic cocktail (Abx) \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e for 5 days prior to 2% DSS \u003cem\u003ead libitum\u003c/em\u003e in drinking water for 6 days. Mice (n\u0026thinsp;=\u0026thinsp;6) without Abx pre-treatment was used as vehicle control. Fecal samples from days 3 and 5 were processed by mixing 0.05 g of fresh feces with 500 \u0026micro;L PBS, vortexed for 3 min, and centrifuged at 14,000\u0026times;g for 10 min. The supernatant was used for DSS quantification as described in DSS quantification section. For E. coli gavage experiment, mice received Abx-water were subsequently administered \u003cem\u003eE. coli\u003c/em\u003e wild type and mutant strains \u003cem\u003evia\u003c/em\u003e oral gavage at a dose of 1.0 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e cfu/200 \u0026micro;L each. DSS was supplied in drinking water at 3% (w/v) for 6 days. Caecum was harvested for sulfite quantification as described in \u003cspan refid=\"Sec16\" class=\"InternalRef\"\u003eSulfite Quantification\u003c/span\u003e section.\u003c/p\u003e \u003cp\u003eMice were monitored daily for body weight, stool consistency and stool bleeding. Mice were euthanized by cervical dislocation after 5\u0026ndash;6 days of DSS treatment. Colon samples were collected for histological, western blot and qRT-PCR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBacterial H\u003csub\u003e2\u003c/sub\u003eS Production from λ-Carrageenan and DSS\u003c/h2\u003e \u003cp\u003eBacteria from the LB start culture were harvested, washed, and inoculated in 5 mL sulfur-free M9 medium with 1% λ-carrageenan (inoculum size 1:200, \u003cem\u003ev/v\u003c/em\u003e), incubated overnight at 37\u0026deg;C while a suspended lead acetate strip monitored H\u003csub\u003e2\u003c/sub\u003eS generation. Medium without inoculum was set up as a negative control.\u003c/p\u003e \u003cp\u003eTo assess DSS degradation, mouse and human fecal samples (2 CD subjects and 2 healthy individuals) were cultured in 10 mL LB medium (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0). Bacterial pellets were resuspended in 5 mL sulfur-free M9 medium with 1% DSS. DSS levels in supernatants were measured via SEC-HPLC, and H\u003csub\u003e2\u003c/sub\u003eS production was monitored with suspended lead acetate strips.\u003c/p\u003e \u003cp\u003eTo investigate H\u003csub\u003e2\u003c/sub\u003eS production from DSS by E. coli WT, mutants, and \u003cem\u003eP. mirabilis\u003c/em\u003e, cultures were grown overnight in 10 mL LB medium at 37\u0026deg;C, 250 rpm. After harvesting, cells were resuspended in 1 mL sulfur-free M9 medium. Pre-incubated DSS medium was prepared using supernatant from M9 medium with 1% DSS and cultures from two CD subjects, as described earlier. Subsequently, 200 \u0026micro;L of this pre-incubated DSS medium and sulfur-free M9 medium with 1% DSS were inoculated with 50 \u0026micro;L of bacterial cultures. Incubation was conducted at 37\u0026deg;C in a 96-well plate with lead acetate strips for sulfide quantification (see Sulfide Quantification, Lead Acetate Strip section).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed with Prism v.8.0 (GraphPad). For two-group comparisons, the statistical significance was determined by unpaired t test or nonparametric Mann-Whitney test as indicated. Multiple group comparisons were made by ANOVA for most of the studies as indicated. Each data point denotes individual human subject, animal, or biological replicate.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy research protocols were reviewed and approved by the Ethical Committees of the First Affiliated Hospital of Sun Yat-sen University (2016[113]). Written informed consent was obtained from all participants. All animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the First Affiliated Hospital of Sun Yat-sen University (2021 [303], 2023 [183]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the National Natural Science Foundation of China (82100577 to Y.Z., 82270579 to R. F., 82370551 to M. C.), Natural Science Foundation of Guangdong Province (2021A1515010572 to R.F.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll study data are included in the article and/or SI Appendix. Data are available in a public, open access repository. Gene expression profiling by high-throughput sequencing data have been deposited in Gene Expression Omnibus accession no. (GSE83687 and GSE131032) and Biostudies accession no. (E-MTAB-54674). Metagenomic sequences for the PRISM is available via SRA with BioProject number PRJNA400072. Raw metagenomic data of the FAH-SYS cohort were deposited in the NCBI public repository (Bioproject #PRJNA793776). HMP IBD metagenomics and transcriptomic data can be accessed at https://ibdmdb.org/tunnel/public/summary.html. All plasmids, bacterial mutant strains, and reagents generated in this study are available from the lead contact upon completing Material Transfer Agreement. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.C., M.D., R. F., and Y.Z. designed research; W.L., M.Z., W.X., R.F., S.X., X.L., L.X., X.L, and Y. G. performed research; W.L., M.Z., M.D. and X.L. analyzed data; and M.D. M.H.M, H.S., and Y.Z. wrote the paper.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the First Affiliated Hospital of Sun Yat-sen University Research Computing for computational resources, maintenance, and support. We thank the First Affiliated Hospital of Sun Yat-sen University Mass Spectrometry Core Laboratory, especially Yuanmei Ye, for their assistance with mass spectrometry analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBurisch, J., \u003cem\u003eet al.\u003c/em\u003e Health-care costs of inflammatory bowel disease in a pan-European, community-based, inception cohort during 5 years of follow-up: a population-based study. Lancet Gastroenterol Hepatol 5, 454\u0026ndash;464 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTargownik, L.E., \u003cem\u003eet al.\u003c/em\u003e Longitudinal Trends in the Direct Costs and Health Care Utilization Ascribable to Inflammatory Bowel Disease in the Biologic Era: Results From a Canadian Population-Based Analysis. 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Cell 180, 862\u0026ndash;877 e822 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Inflammatory bowel disease, inorganic sulfate, sulfopolysaccharide, 3’-Phosphoadenosine-5’-phosphosulfate PAPS, Adenosine-5’-phosphosulfate APS","lastPublishedDoi":"10.21203/rs.3.rs-4176488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4176488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS imbalances in the intestinal tract trigger Crohn's disease (CD), a chronic inflammatory gastrointestinal disorder characterized by microbiota dysbiosis and barrier dysfunction. However, a comprehensive understanding of H\u003csub\u003e2\u003c/sub\u003eS generation in the gut, and the contributions of both microbiota and host to systemic H\u003csub\u003e2\u003c/sub\u003eS levels in CD, remain to be elucidated. This investigation aimed to enhance comprehension regarding the sulfidogenic potential of both the human host and the gut microbiota.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur analysis of a treatment-naive CD cohorts' fecal metagenomic and biopsy metatranscriptomic data revealed reduced expression of host endogenous H\u003csub\u003e2\u003c/sub\u003eS generation genes alongside increased abundance of microbial exogenous H\u003csub\u003e2\u003c/sub\u003eS production genes in correlation with CD. While prior studies focused on microbial H\u003csub\u003e2\u003c/sub\u003eS production \u003cem\u003evia\u003c/em\u003e dissimilatory sulfite reductases, our metagenomic analysis suggests the assimilatory sulfate reduction (ASR) pathway is a more significant contributor in the human gut, given its high prevalence and abundance. Subsequently, we validated our hypothesis experimentally by generating ASR-deficient \u003cem\u003eE. coli\u003c/em\u003e mutants \u003cem\u003e∆cysJ\u003c/em\u003e and \u003cem\u003e∆cysM\u003c/em\u003e through the deletion of sulfite reductase and L-cysteine synthase genes. This alteration significantly affected bacterial sulfidogenic capacity, colon epithelial cell viability, and colonic mucin sulfation, ultimately leading to colitis in murine model. Further study revealed that gut microbiota degrade sulfopolysaccharides and assimilate sulfate to produce H\u003csub\u003e2\u003c/sub\u003eS \u003cem\u003evia\u003c/em\u003e the ASR pathway, highlighting the role of sulfopolysaccharides in colitis and cautioning against their use as food additives.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur study significantly advances understanding of microbial sulfur metabolism in the human gut, elucidating the complex interplay between diet, gut microbiota, and host sulfur metabolism. We highlight the microbial ASR pathway as an overlooked endogenous H\u003csub\u003e2\u003c/sub\u003eS producer and a potential therapeutic target for managing CD.\u003c/p\u003e","manuscriptTitle":"Microbial Assimilatory Sulfate Reduction-Mediated H2S: An Overlooked Role in Crohn's Disease Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-02 16:43:07","doi":"10.21203/rs.3.rs-4176488/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-11T15:28:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-11T15:27:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-28T22:48:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbiome","date":"2024-03-27T13:33:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"64887d0d-ce24-4bdc-9dd3-9f562c412c83","owner":[],"postedDate":"April 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-22T19:35:32+00:00","versionOfRecord":{"articleIdentity":"rs-4176488","link":"https://doi.org/10.1186/s40168-024-01873-2","journal":{"identity":"microbiome","isVorOnly":false,"title":"Microbiome"},"publishedOn":"2024-08-16 15:57:33","publishedOnDateReadable":"August 16th, 2024"},"versionCreatedAt":"2024-04-02 16:43:07","video":{"identity":"a455da376f8666d416be4ef9f5ab1f1f"},"vorDoi":"10.1186/s40168-024-01873-2","vorDoiUrl":"https://doi.org/10.1186/s40168-024-01873-2","workflowStages":[]},"version":"v1","identity":"rs-4176488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4176488","identity":"rs-4176488","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}