Abstract:Some well-known Clostridiales species such as
Clostridium difficile
and
C. perfringens
are agents of high impact diseases worldwide. Nevertheless, other foreseen Clostridiales species have recently emerged such as
Clostridium tertium
and
C. paraputrificum
. Three fecal isolates were identified as
Clostridium tertium
(Gcol.A2 and Gcol.A43) and
C. paraputrificum
(Gcol.A11) dur… Show more
“…C. disporicum is a saccharolytic bacterium that presents epimerase activity resulting in the production of ursodeoxycholic acid 24,25 . C. tertium is an uncommon pathogen, albeit associated with cases of severe diseases, mostly in immunocompromised and neutropenic patients 26 . P. benzoelyticum can ferment some amino acids, including threonine, a major component of mucin backbone 27 .…”
Mucins are large glycoproteins consisting of approximately 80% of hetero-oligosaccharides. Gut mucin degraders of healthy subjects were investigated, through a culture dependent and independent approach. The faeces of five healthy adults were subjected to three steps of anaerobic enrichment in a medium with sole mucins as carbon and nitrogen sources. The bacterial community was compared before and after the enrichment by 16S rRNA gene profiling. Bacteria capable of fermenting sugars, such as Anaerotruncus, Holdemania, and Enterococcaceae likely took advantage of the carbohydrate chains. Escherichia coli and Enterobacteriaceae, Peptococcales, the Coriobacteriale Eggerthella, and a variety of Clostridia such as Oscillospiraceae, Anaerotruncus, and Lachnoclostridium, significantly increased and likely participated to the degradation of the protein backbone of mucin. The affinity of E. coli and Enterobacteriaceae for mucin may facilitate the access to the gut mucosa, promoting gut barrier damage and triggering systemic inflammatory responses. Only three species of strict anaerobes able to grow on mucin were isolated from the enrichments of five different microbiota: Clostridium disporicum, Clostridium tertium, and Paraclostridium benzoelyticum. The limited number of species isolated confirms that in the gut the degradation of these glycoproteins results from cooperation and cross-feeding among several species exhibiting different metabolic capabilities.
“…C. disporicum is a saccharolytic bacterium that presents epimerase activity resulting in the production of ursodeoxycholic acid 24,25 . C. tertium is an uncommon pathogen, albeit associated with cases of severe diseases, mostly in immunocompromised and neutropenic patients 26 . P. benzoelyticum can ferment some amino acids, including threonine, a major component of mucin backbone 27 .…”
Mucins are large glycoproteins consisting of approximately 80% of hetero-oligosaccharides. Gut mucin degraders of healthy subjects were investigated, through a culture dependent and independent approach. The faeces of five healthy adults were subjected to three steps of anaerobic enrichment in a medium with sole mucins as carbon and nitrogen sources. The bacterial community was compared before and after the enrichment by 16S rRNA gene profiling. Bacteria capable of fermenting sugars, such as Anaerotruncus, Holdemania, and Enterococcaceae likely took advantage of the carbohydrate chains. Escherichia coli and Enterobacteriaceae, Peptococcales, the Coriobacteriale Eggerthella, and a variety of Clostridia such as Oscillospiraceae, Anaerotruncus, and Lachnoclostridium, significantly increased and likely participated to the degradation of the protein backbone of mucin. The affinity of E. coli and Enterobacteriaceae for mucin may facilitate the access to the gut mucosa, promoting gut barrier damage and triggering systemic inflammatory responses. Only three species of strict anaerobes able to grow on mucin were isolated from the enrichments of five different microbiota: Clostridium disporicum, Clostridium tertium, and Paraclostridium benzoelyticum. The limited number of species isolated confirms that in the gut the degradation of these glycoproteins results from cooperation and cross-feeding among several species exhibiting different metabolic capabilities.
“…These sequences are encoded by C. paraputrificum s trains isolated from preterm infants, namely strain LH025, LH141, and LH058, 23 or isolated from feces (Gcol.A11). 24 Figure 5. Maximum-likelihood tree based on a subset of the taxa present in the full phylogenetic analysis of 12β-HSDH and SDS-PAGE of proteins explored further.…”
Section: Resultsmentioning
confidence: 99%
“… 13 , 35 , 41 7α- and 7β-HSDH were shown in numerous Firmicutes 14 , 37 , 65 and the Actinobacteria Collinsella aerofaciens . 24 Along with these bile acid-specific HSDHs, the glucocorticoid 20α- and 20β-HSDHs are evident in both Firmicutes 43 , 45 and Actinobacteria such as Bifidobacterium adolescentis. 42 , Until this study, there were no reports of genes encoding 12β-HSDH and the activity had only been shown in C. paraputrificum, C. tertium and C. difficile .…”
Bile acids are detergent molecules that solubilize dietary lipids and lipid-soluble vitamins. Humans synthesize bile acids with α-orientation hydroxyl groups which can be biotransformed by gut microbiota to toxic, hydrophobic bile acids, such as deoxycholic acid (DCA). Gut microbiota can also convert hydroxyl groups from the α-orientation through an oxo-intermediate to the β-orientation, resulting in more hydrophilic, less toxic bile acids. This interconversion is catalyzed by regio- (C-3 vs. C-7) and stereospecific (α vs. β) hydroxysteroid dehydrogenases (HSDHs). So far, genes encoding the urso- (7α-HSDH & 7β-HSDH) and iso- (3α-HSDH & 3β-HSDH) bile acid pathways have been described. Recently, multiple human gut clostridia were reported to encode 12α-HSDH, which interconverts DCA and 12-oxolithocholic acid (12-oxoLCA). 12β-HSDH completes the epi-bile acid pathway by converting 12-oxoLCA to the 12β-bile acid denoted epiDCA; however, a gene(s) encoding this enzyme has yet to be identified. We confirmed 12β-HSDH activity in cultures of
Clostridium paraputrificum
ATCC 25780. From six candidate
C. paraputrificum
ATCC 25780 oxidoreductase genes, we discovered the first gene (DR024_RS09610) encoding bile acid 12β-HSDH. Phylogenetic analysis revealed unforeseen diversity for 12β-HSDH, leading to validation of two additional bile acid 12β-HSDHs through a synthetic biology approach. By comparison to a previous phylogenetic analysis of 12α-HSDH, we identified the first potential C-12 epimerizing strains:
Collinsella tanakaei
YIT 12063 and
Collinsella stercoris
DSM 13279. A Hidden Markov Model search against human gut metagenomes located putative 12β-HSDH genes in about 30% of subjects within the cohorts analyzed, indicating this gene is relevant in the human gut microbiome.
“…These sequences are encoded by C. paraputrificum s trains isolated from preterm infants, namely strain LH025, LH141, and LH058 23 , or isolated from feces (Gcol.A11). 24…”
Section: Resultsmentioning
confidence: 99%
“…Cp12β-HSDH clustered most closely with other C. paraputrificum sequences (WP_099327725, WP_049179624, WP_111937163-). These sequences are encoded by C. paraputrificum strains isolated from preterm infants, namely strain LH025, LH141, and LH058 (28), or isolated from feces (Gcol.A11) (29).…”
Section: Phylogenetic Analysis Of Cp12β-hsdhmentioning
Bile acids are detergent molecules that solubilize dietary lipids and lipid-soluble vitamins. Humans synthesize bile acids with α-orientation hydroxyl groups which can be biotransformed by gut microbiota to toxic, hydrophobic bile acids, such as deoxycholic acid (DCA). Gut microbiota are also capable of converting hydroxyl groups from the α-orientation through an oxo-intermediate to the β-orientation, resulting in more hydrophilic and less toxic bile acids. This interconversion is catalyzed by regio- (C-3 vs. C-7) and stereospecific (α vs. β) hydroxysteroid dehydrogenases (HSDHs). Recently, multiple human gut clostridia have been reported to encode 12α-HSDH, which interconverts DCA and 12-oxolithocholic acid (12-oxoLCA). Bile acid 12β-HSDH activity completes the epimerization of DCA by converting 12-oxoLCA to the 12β-bile acid known as epiDCA. While 12β-HSDH activity has been shown in cell extracts of Clostridium paraputrificum, the gene has not yet been reported. In order to identify the first gene encoding this activity, 6 candidate oxidoreductase genes from C. paraputrificum ATCC 25780 were cloned, overexpressed, purified, and screened for activity with 12-oxoLCA and epiDCA. LC-MS analysis was performed on reaction products from the enzyme encoded by DR024_RS09610, confirming the first 12β-HSDH gene discovered. The enzyme was more specific for bile acids lacking a 7-hydroxyl group than cholic acid derivatives containing a 7-hydroxyl. Phylogenetic analysis revealed previously unknown diversity for bile acid 12β-HSDH by experimentally validating two additional 12β-HSDHs within the tree from Eisenbergiella sp. OF01-20 and Olsenella sp. GAM18.
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