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Title:Microbial pathways of sulfur metabolism and colorectal cancer risk
Author(s):Wolf, Patricia G.
Director of Research:Gaskins, H. Rex
Doctoral Committee Chair(s):Donovan, Sharon M
Doctoral Committee Member(s):Ridlon, Jason M; Mackie, Roderick I; Miller, Michael J
Department / Program:Nutritional Sciences
Discipline:Nutritional Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Sulfur metabolism
Colorectal cancer
Sulfur Amino Acids
Sulfide
Sulfidogenic bacteria
Bilophila wadsworthia
SRB
Sulfate reducing bacteria
Taurine
Cysteine
Abstract:Colorectal cancer (CRC) is the third leading cause of cancer, and the second leading cause of cancer death in the United States (US). 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 (H2S), produced by resident sulfidogenic bacteria, triggers hyper-proliferation and pro-inflammatory pathways, and is genotoxic. In the US, there is a higher incidence of CRC in African Americans (AAs) compared to non-Hispanic whites (NHWs). We hypothesized that sulfidogenic bacterial abundance in colonic mucosa may be an environmental CRC risk factor that distinguishes AAs 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. African Americans 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 (SRB), dissimilatory sulfate reductase (pan-dsrA), Bilophila wadsworthia-specific dsrA, and 16S rRNA genes for Desulfobacter spp., Desulfovibrio spp., and Desulfotomaculum spp.. Bilophila wadsworthia-specific dsrA was 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 features that characterize AA CRC. Fat intake and daily servings of meat were higher (p<0.01) in AAs compared with NHWs, and dietary fat intake correlated positively with pan-dsrA abundance (p=0.011). Additionally, dairy and calcium intake were 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. Sulfidogenic bacteria metabolize organic and inorganic sulfur in order to produce H2S. We observed that microbes that metabolize organic sulfur distinguished AA CRC from NHW CRC subjects and AA controls. We hypothesize that SRB, (inorganic sulfur metabolizers), may impart beneficial functions, such as hydrogen disposal and barrier protection through antimicrobial effects of sulfide. Increased abundance of bacteria that utilize organic sources of sulfur may increase sulfide concentrations to levels that are proinflammatory and genotoxic. Bilophila wadsworthia produces H2S through metabolism of taurine, a sulfur amino acid available in the colon by diet and taurine conjugated bile acids. While studies have demonstrated preference for taurine as a terminal electron acceptor by B. wadworthia in vitro, none have determined the in vivo metobolic ‘lifestyle’ in a defined mixed community. Thus, as it was one of the most significant markers distinguishing AA CRC, we aimed to observe the baseline metabolic activity of this bacterium in vivo in mice fed a standard chow diet. Six gnotobiotic mice were colonized with a synthetic microbial community previously validated to be capable of metabolizing the taurine conjugated bile acid taurocholate (TCA), and were housed at the Mayo Clinic Gnotobiotic Facility for 30 days. A transcriptome analysis of cecal content revealed 4099 transcripts expressed by B. wadsworthia. Transcripts were observed for all three enzymes involved in taurine metabolism: taurine:pyruvate aminotransferase (tpa), sulfoacetaldehyde sulfolyase, and dissimilatory sulfite reductase A (dsrA). Taurine metabolism was the most abundant metabolic pathway expressed by the microbe, and dsrA an upstream enzyme to tpa were among the most abundant genes transcribed. Community analysis revealed expression of genes involved in liberating taurine from the bile acid taurocholate. Additionally, genes for D-cysteine and nitrogen metabolism were highly expressed, indicating that alternate forms of metabolism may be important for pathogenesis and microbial fitness. These results indicate that B. wadsworthia is likely producing H2S in the colonic environment from taurine and D-cysteine metabolism, and that taurine from TCA metabolism is an available substrate for this microbe. In addition to B. wadsworthia, our recent study revealed Odoribacter spp. as a feature that distinguished AA CRC. Therefore, we aimed to verify that members of this genus do produce sulfide, and to determine the mechanism of their sulfur metabolism. The microbial species Odoribacter splanchnicus served as a surrogate for this analysis. Culture of O. splanchnicus in Sulfide Indole and Motility Medium revealed the microbe does indeed produce H2S. A genome search revealed a bifunctional tryptophanase/cysteine desulfhydrase (tnaA). The gene tnaA from O. splanchnicus was amplified by PCR, ligated into the expression vector pET-28a, transformed into Eschericia coli strain BL21, and overexpressed at 16°C for 16 hours. Functional analysis of the recombinant enzyme revealed positive cysteine desulfhydrase and tryptophanase activity. Cysteine desulfhydrase activity resulted in the consumption of cysteine and production of H2S and pyruvate. This implicates tnaA as an important functional enzyme in future analyses of the microbiome in CRC. Given that our analyses revealed microbial organic sulfur metabolism as a significant indicator of CRC, and identified two additional sulfidogenic enzymes to be considered in evaluations of sulfur metabolism in the human gut, an examination of the sulfidogenic capacity of the human microbiome was performed. A survey of genomes published from the Human Microbiome Project (HMP) revealed that over a third of microbial species harbored genes for sulfur metabolism. Genes for cysteine metabolism were the most abundant sulfidogenic genes, revealing that it may be a more important driver of sulfidogenesis than inorganic sulfate metabolism in the human gut. In addition, a retroactive analysis revealed several species that were significantly correlated with CRC harbored genes for sulfur metabolism. These findings suggest that sulfur metabolism is more wide-spread in the human gut than previously understood, and that H2S production from organic sulfur metabolism may be an environmental trigger of CRC.
Issue Date:2018-12-06
Type:Text
URI:http://hdl.handle.net/2142/102846
Rights Information:Copyright 2018 Patricia G. Wolf
Date Available in IDEALS:2019-02-07
Date Deposited:2018-12


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