Chapter Four - Role of sulfidogenic members of the gut microbiota in human disease

Objectives. Colorectal cancer (CRC) incidence is higher in post-industrial cultures, and the incidences varies among different populations. In the US, there is a higher incidence of CRC in African Americans (AAs) compared to non-Hispanic whites (NHWs). Recent evidence links consumption of a diet high in animal protein and fat as an environmental risk factor for the development of CRC. The intestinal microbiota is postulated to modulate the effects of diet in promoting or preventing CRC development. Hydrogen sulfide, which is produced by normal members of the colonic microenvironment (sulfidogenic bacteria), triggers pro-inflammatory and hyper-proliferative pathways, and it is genotoxic. We hypothesized that the production of hydrogen sulfide by sulfidogenic bacteria is a key environmental carcinogen contributing to CRC risk. Design. The abundance of sulfidogenic bacteria via quantitative PCR (qPCR) was compared in non-involved colonic mucosa of 97 AA and 56 NHW CRC patients and in 100 AA and 76 NHW healthy controls. In addition, we performed 16S rDNA sequencing in 61 AA cases and 94 AA controls. Additionally, we tested correlations among race, dietary intake, disease status, and sulfidogenic bacterial abundance. Results. Overall, the functional gene for hydrogen sulfide production in sulfate-reducing bacteria, dissimilatory sulfate reductase (dsrA), was more abundant in AAs than in NHWs, in both cases and controls. In addition, AA CRC cases exhibited a significantly higher abundance of Bilophila wadsworthia-specific dsrA. Linear discriminant analysis of 16S rDNA sequencing results revealed several taxa that differed between AA cases and controls, including the known butyrate producer Faecalibacterium that was more abundant in AA controls, and the sulfidogenic Pyramidobacter that was more abundant in AA CRC cases. Importantly, we found that dietary intake of protein and fat was higher in AAs compared to NHWs, and these dietary components correlated with a higher abundance of sulfidogenic bacteria. Conclusion. There were significant differences in sulfidogenic bacterial abundance between AAs and NHWs, in both cases and controls, and implicate sulfidogenic bacteria as an important diet-driven environmental exposure that contributes to the increased risk of CRC in AAs. Replication studies are needed to test that effectiveness of using B. wadsworthia as a biomarker for increased CRC risk. Citation Format: Cemal Yazici, Patricia G. Wolf, Tzu-Wen Liu, Karin Vermillion, Timothy Carroll, Ece Mutlu, Lisa Tussing-Humphreys, Carol Braunschweig, Rosa M. Xicola, Barbara Jung, Xavier Llor, Nathan A. Ellis, H. Rex Gaskins. Sulfidogenic bacteria are an important diet-driven exposure promoting colorectal cancer in African Americans. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B31.

Colorectal cancer (CRC) is one of the most common and fatal malignant tumors globally, and its development is increasingly related to the gut microbiota. Despite its effect on CRC having been extensively researched, the intestinal mucus barrier, which forms a fundamental link between the host tissues and gut microbes, is seldom discussed. A double-layered barrier, mainly formed by MUC2 mucin, isolates the outside world from epithelial cells to maintain intestinal homeostasis. Furthermore, it is subjected to a dynamic impact of microbial activity. Now, increasing evidence shows that mucus barrier disruption driven by certain gut microbes is an early event in the development of CRC. This review first introduces the structure and function of the colonic mucus barrier and then discusses how gut microbiota in different areas promote the development of CRC by disrupting the mucus layer. Finally, we examine translational opportunities for exploiting microbiota–mucus barrier interactions in CRC therapy.

Akkermansia muciniphila in inflammatory bowel disease and colorectal cancer.

Oral delivery of ultra-small zwitterionic nanoparticles to overcome mucus and epithelial barriers for macrophage modulation and colitis therapy.

The gut microbiota is a complex ecosystem that plays a critical role in human health and disease. However, the relationship between gut microbiota and intestinal damage caused by burns is not well understood. The intestinal mucus layer is crucial for maintaining intestinal homeostasis and providing a physiological barrier against bacterial invasion. This study aims to investigate the impact of gut microbiota on the synthesis and degradation of intestinal mucus after burns and explore potential therapeutic targets for burn injury. A modified histopathological grading system was employed to investigate the effects of burn injury on colon tissue and the intestinal mucus barrier in mice. Subsequently, 16S ribosomal RNA sequencing was used to analyze alterations in the gut microbiota at days 1-10 post-burn. Based on this, metagenomic sequencing was conducted on samples collected at days 1, 5 and 10 to investigate changes in mucus-related microbiota and explore potential underlying mechanisms. Our findings showed that the mucus barrier was disrupted and that bacterial translocation occurred on day 3 following burn injury in mice. Moreover, the gut microbiota in mice was significantly disrupted from days 1 to 3 following burn injury, but gradually recovered to normal as the disease progressed. Specifically, there was a marked increase in the abundance of symbiotic and pathogenic bacteria associated with mucin degradation on day 1 after burns, but the abundance returned to normal on day 5. Conversely, the abundance of probiotic bacteria associated with mucin synthesis changed in the opposite direction. Further analysis revealed that after a burn injury, bacteria capable of degrading mucus may utilize glycoside hydrolases, flagella and internalins to break down the mucus layer, while bacteria that synthesize mucus may help restore the mucus layer by promoting the production of short-chain fatty acids. Burn injury leads to disruption of colonic mucus barrier and dysbiosis of gut microbiota. Some commensal and pathogenic bacteria may participate in mucin degradation via glycoside hydrolases, flagella, internalins, etc. Probiotics may provide short-chain fatty acids (particularly butyrate) as an energy source for stressed intestinal epithelial cells, promote mucin synthesis and accelerate repair of mucus layer.

In the US, there is a higher incidence of colorectal cancer (CRC) in African Americans (AAs) compared to non‐Hispanic whites (NHWs). Recent evidence links consumption of a diet high in animal protein and fat as an environmental risk factor for CRC development, and the intestinal microbiota modulates the tumor promoting or protective effects of diet. Hydrogen sulfide, produced by resident sulfidogenic bacteria, triggers pro‐inflammatory pathways and hyper‐proliferation, and is genotoxic. We hypothesized that sulfidogenic bacterial abundance in colonic mucosa may be an environmental CRC risk factor that distinguishes AA and NHWs, and may be correlated with differences in dietary composition. Colonic biopsies from uninvolved or healthy mucosa from CRC cases and controls were collected from five medical centers through the Chicago Colorectal Cancer Consortium. Using quantitative PCR, sulfidogenic bacterial abundance was measured in uninvolved colonic mucosa of 97 AA and 56 NHW CRC cases, and 100 AA and 76 NHW controls. In addition, 16S rRNA sequencing was performed in AA cases and AA controls. A Block Brief 2000 Food Frequency Questionnaire was collected from a subset of subjects of 50 AA and 31 NHW CRC cases and 30 AA and 24 NHW controls. Differences were examined among bacterial targets, race, disease status, and dietary intake. AAs harbored a greater abundance (p<0.001) of sulfidogenic bacteria compared to NHWs regardless of disease status, including the functional gene for H2S production in sulfate reducing bacteria, dissimilatory sulfate reductase (pan‐dsrA), as well as Bilophila wadsworthia‐specific dsrA, and 16S rRNA genes for Desulfobacter spp., Desulfovibrio spp., and Desulfotomaculum spp. Bilophila wadsworthia‐specific dsrA was significantly more abundant in AA cases compared to AA controls (p<0.001). Linear discriminant analysis of 16S rRNA gene sequences highlighted the sulfidogenic Bilophila, Lactococcus, Odoribacter, Porphyromonas and Pyramidobacter genera as significant in AA cases. Fat intake and daily servings of meat were significantly higher (p<0.01) in AAs compared with NHWs, and dietary fat intake correlated positively with pan‐dsrA abundance (p=0.011). Additionally, intake of dairy and calcium was lower (p<0.001) in AA, and servings of dairy correlated negatively with pan‐dsrA abundance (p=0.007). Together, these results implicate sulfidogenic bacteria as an environmental risk factor contributing to CRC development in AAs.Support or Funding InformationThis work was supported by grants from the National Cancer Institute (U01 CA153060 and P30 CA023074, NAE; RO1 CA204808, HRG, EM, LTH; RO1 CA141057, BJ) and the American Cancer Society Illinois Division (223187, XL). PGW was supported by a predoctoral fellowship from Mayo Clinic and University of Illinois Alliance for Technology‐Based Healthcare. CY was supported by a training grant from National Institute of Health (5T32DK007788‐15). GJA was supported by a Cancer Biology Training Grant (T32CA009213).

Intestinal mucus barrier dysfunction is closely involved in the pathogenesis of inflammatory bowel diseases (IBD). To investigate the protective effect and underlying mechanism of arctigenin, a phytoestrogen isolated from the fruits of Arctium lappa L., on the intestinal mucus barrier under colitis condition. The role of arctigenin on the intestinal mucus barrier and the apoptosis of goblet cells were examined by using both in vitro and in vivo assays. Arctigenin was demonstrated to promote the mucus secretion and maintain the integrity of mucus barrier, which might be achieved by an increase in the number of goblet cells via inhibiting apoptosis. Arctigenin selectively inhibited the mitochondrial pathway-mediated apoptosis. Moreover, arctigenin elevated the protein level of prohibitin 1 (PHB1) through blocking the ubiquitination via activation of estrogen receptor β (ERβ) to competitively interact with PHB1 and disrupt the binding of tripartite motif 21 (TRIM21) with PHB1. ERβ knock down in the colons of mice with DSS-induced colitis resulted in significant reduction of the protection of arctigenin and DPN against the mucosal barrier. Arctigenin can maintain the integrity of the mucus barrier by inhibiting the apoptosis of goblet cells through the ERβ/TRIM21/PHB1 pathway.

A comprehensive understanding of how dietary components impact immunoregulatory gene expression in adipose tissue (AT) and liver, and their respective contributions to metabolic health in mice, remains limited. The current study aimed to investigate the metabolic consequences of a high-sucrose diet (HSD) and a high-fat diet (HFD) in female mice with a focus on differential lipid- and sucrose-induced changes in immunoregulatory gene expression in AT and liver. Female C57BL/6 J mice were fed a purified and macronutrient matched high fat, high sugar, or control diets for 12 weeks. Mice were extensively phenotyped, including glucose and insulin tolerance tests, adipose and liver gene and protein expression analysis by qPCR and Western blot, tissue lipid analyses, as well as histological analyses. Compared to the control diet, HSD- and HFD-fed mice had significantly higher body weights, with pronounced obesity along with glucose intolerance and insulin resistance only in HFD-fed mice. HSD-fed mice exhibited an intermediate phenotype, with mild metabolic deterioration at the end of the study. AT lipid composition was significantly altered by both diets, and inflammatory gene expression was only significantly induced in HFD-fed mice. In the liver however, histological analysis revealed that both HSD- and HFD-fed mice had pronounced ectopic lipid deposition indicating hepatic steatosis, but more pronounced in HSD-fed mice. This was in line with significant induction of pro-inflammatory gene expression specifically in livers of HSD-fed mice. Overall, our findings suggest that HFD consumption in female mice induces more profound inflammation in AT with pronounced deterioration of metabolic health, whereas HSD induced more pronounced hepatic steatosis and inflammation without yet affecting glucose metabolism.

Oral drug delivery systems had natural potential for colorectal cancer drug therapy. While the drug delivery efficiency is severely hindered by the complex intestinal barriers, especially mucus and epithelium barriers, resulting in unsatisfactory therapeutic effects and limited clinical translation. In this work, a bioactive self-thermophoretic and gas dual-driven nanomotor is developed for colorectal cancer therapy through efficient intestinal mucus and epithelial barrier penetration. The nanomotor shows intestinal mucus barrier penetration and the paracellular pathway reversibly opening properties of intestinal epithelium barrier, increasing the delivery efficiency of cisplatin by 3.5 folds. Owing to the targeted delivery of cisplatin and the reduced side effects on normal intestinal tissues, the therapeutic efficiency of the nanomotor for colorectal cancer in vivo is as high as 98.6%. With autonomous and reversible intestinal barriers penetration property, the nanoplatform may innovate the current oral drug delivery.

The present study aimed to determine if metformin exerts anti-inflammatory and mucus-protective effects via the gut microbiota. Metformin has extensive benefits including anti-inflammatory effects. Previous studies showed that metformin changed the gut microbiota composition and increases the number of goblet cells. Intestinal dysbiosis and goblet cell depletion are important features of ulcerative colitis (UC). The underlying mechanism and whether metformin can improve the mucus barrier in UC remain unclear. Metformin (400 mg/kg/day) was administered to mice with dextran sulfate sodium (DSS)-induced UC for 2 wk to investigate the effects of metformin on the intestinal mucus barrier. The gut microbiota was depleted, using antibiotics, to explore its role in the mucus-protecting effects of metformin. Akkermansia muciniphila (A. muciniphila), which was enriched in metformin-treated mice, was administered to mice to investigate the effects of the bacteria on UC and the mucus barrier. Metformin attenuated DSS-induced UC in mice, as evidenced by the alleviation of diarrhea, hematochezia, and the decrease in body weight. The expression of mucin2, a prominent mucus barrier protein, was increased in the metformin-treated group compared to the DSS-treated group. Furthermore, fecal 16S rRNA analysis showed that metformin treatment changed the gut microbiota composition by increasing the relative abundance of Lactobacillus and Akkermansia species while decreasing Erysipelatoclostridium at the genus level. Antibiotic treatment partly abolished the anti-inflammatory and mucus-protecting effects of metformin. Administration of A. muciniphila alleviated the colonic inflammation and mucus barrier disruption. Metformin alleviated DSS-induced UC in mice and protected against cell damage via affecting the gut microbiota, thereby providing a new mechanism for the therapeutic effect of metformin in patients with UC. This study also provides evidence that A. muciniphila as a probiotic has potential benefits for UC.

Upregulated Tβ4 expression in inflammatory bowel disease impairs the intestinal mucus barrier by inhibiting autophagy in mice

ObjectiveColorectal cancer (CRC) incidence is higher in African Americans (AAs) compared with non-Hispanic whites (NHWs). A diet high in animal protein and fat is an environmental risk factor for CRC...

The proinflammatory and genotoxic properties of hydrogen sulfide (H2S) have been implicated as an environmental trigger of colonic disease. Recent evidence links sulfidogenic bacteria capable of metabolizing organic sulfur substrates, like dietary sulfur amino acids and taurine conjugated bile acids, with increased colorectal cancer (CRC) and colitis risk as a consequence of a “western” type diet. Our previous work demonstrates that the sulfidogenic bacterium, Odoribacter splanchnicus, is a significant indicator of CRC in African Americans who are at higher risk of CRC incidence and death than other races and ethnicities in the United States. This observation has been corroborated by several recent studies that observed O. splanchnicus to be associated with CRC and colitis-induced CRC. While previous work has reported that O. splanchnicus may be sulfidogenic, its method of H2S production has yet to be characterized. Production of H2S by O. splanchnicus was confirmed in vitro using sulfide indole and motility media, and varying sulfur substrates. A predictive metabolic analysis of 74 O. splanchnicus metagenomes taken from publicly available CRC cohorts did not reveal complete pathways commonly associated with H2S production, however genes for a polar amino acid transporter were revealed. Therefore, a genomic analysis was performed revealing that O. splanchnicus harbors the sulfidogenic genes tryptophanase (tnaA) and cystathionine-β-synthase. The enzyme TnaA has been previously shown in Eschericia coli to produce indole or H2S from tryptophan or cysteine, respectively. However, the E. coli amino acid sequence shares only 44.85% identity with that from O. splanchnicus, thus heterologous protein expression of recombinant tnaA was performed to determine enzyme functionality. Enzyme kinetics and pH optimum were obtained using the spectrophotometric BiCl3 assay, which turns black in presence of H2S. Functional analysis of recombinant purified TnaA revealed bifunctionality of the enzyme with positive tryptophanase and cysteine desulfhydrase activity with differing substrates. Cysteine desulfhydrase activity resulted in consumption of L-cysteine with subsequent production of H2S and pyruvate. Thus, TnaA is a functional enzyme that warrants further evaluation as possibly relating to sulfidogenic O. splanchnicus being linked to CRC and colitis-associated CRC. Citation Format: Patricia G. Wolf, Vladimir Kolosslov, Zhichao Zhou, Lindsey Ly, Heidi Doden, Saravanan Devendran, Adam M. Breister, Luke Lucio, Paige Polak, Sarah Matatov, Karthik Anantharaman, Jason M. Ridlon, Rex Gaskins. The colorectal cancer associated microbe Odoribacter splanchnicus produces genotoxic hydrogen sulfide via cysteine metabolism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3342.

The Western diet characterized by high fat and high sugar is considered a risk factor for many inflammatory disorders including colitis. However, the impact of dietary sugars, such as glucose, fructose, and sucrose, on colitis remains poorly understood. We, therefore, investigated the role of dietary simple sugars in colitis using dextran sulfate sodium (DSS) and Il10−/− mice models. We fed WT mice with high sugar in drinking water or diet, followed by administration of 2.5% DSS plus high-sugar diet. Sugar-fed wild-type mice showed extreme sensitivity to DSS-induced colitis. Consistently, Il10−/− mice spontaneously developed severe colitis with the consumption of high sugar. To understand the underlying mechanism of detrimental effect of dietary sugar on colitis, we measured pathophysiological changes in the healthy gut. Interestingly, while there was no induction of cell death, inflammatory mediators, and activation of inflammatory pathways, gut microbiota composition was significantly altered in sugar-fed healthy mice. Notably, the abundance of mucus degrading bacteria Akkermansia muciniphila and Bacteroides fragilis was increased. Consistently, bacteria-derived mucolytic enzymes were enriched and the colonic mucus layers were eroded in sugar-fed mice. Furthermore, germ-free mice colonized with microbiota from sugar-treated mice showed higher abundance of mucus degrading bacteria and increased colitis susceptibility. By demonstrating the complex interplay between high-sugar diet, gut microbiota composition, and intestinal mucus barrier, this study, therefore, elucidates a novel mechanism of colitis pathogenesis.

Nanoparticles (NPs) for oral delivery of peptide/protein drugs are largely limited due to the coexistence of intestinal mucus and epithelial barriers. Sequentially overcoming these two barriers is intractable for a single nanovehicle due to the requirements of different or even contradictory surface properties of NPs. To solve this dilemma, a mucus-penetrating virus-inspired biomimetic NP with charge reversal ability (P-R8-Pho NPs) was developed by densely coating poly(lactic- co-glycolic acid) NPs with cationic octa-arginine (R8) peptide and specific anionic phosphoserine (Pho). The small size (81.81 nm) and viruslike neutral charged surface (-2.39 mV) of the biomimetic NPs achieved rapid mucus penetration, which was almost equal to that of the conventional PEGylated mucus-penetrating nanoparticles. The hydrolysis of surface-anchored anionic Pho was achieved by intestinal alkaline phosphatase, which led to the turnover of ζ potential to positive (+7.37 mV). This timely charge reversal behavior also exposed cationic R8 peptide and induced efficient cell-penetrating peptide (CPP)-mediated cellular uptake and transepithelial transport on Caco-2/E12 cocultured cell model. What's more, P-R8-Pho NPs showed excellent stability in simulated gastrointestinal conditions and enhanced absorption in intestine in vivo. Finally, oral administration of insulin-loaded P-R8-Pho NPs enabled to induce a preferable hypoglycemic effect and a 1.9-fold higher oral bioavailability was achieved compared with single CPP-modified P-R8 NPs on diabetic rats. The combinative application of biomimetic mucus-penetrating strategy and enzyme-responsive charge reversal strategy in a single nanovehicle could sequentially overcome mucus and epithelial barriers, thus showing great potential for the oral peptide/protein delivery.