Bile salt hydrolase acyltransferase activity expands bile acid diversity.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

Longitudinal microbial and molecular dynamics in the cystic fibrosis lung after Elexacaftor-Tezacaftor-Ivacaftor therapy.

BACKGROUND: Cystic fibrosis (CF) is a genetic disorder causing poor mucociliary clearance in the airways and subsequent respiratory infection. The recently approved triple therapy Elexacaftor-Tezacaftor-Ivacaftor (ETI) has significantly improved lung function and decreased airway infection in persons with CF (pwCF). This improvement has been shown to occur rapidly, within the first few weeks of treatment. The effects of longer term ETI therapy on lung infection dynamics, however, remain mostly unknown. RESULTS: Here, we applied 16S rRNA gene amplicon sequencing, untargeted metabolomics, and neutral models to high-resolution, longitudinally collected sputum samples from pwCF on ETI therapy (162 samples, 7 patients) and compared to similarly collected data set from pwCF not taking ETI (630 samples, 9 patients). Because ETI reduces sputum production, samples were collected in freezers provided in the subject's homes at least 3 months after first taking ETI, with those on ETI collecting a sample approximately weekly. The lung function (%ppFEV1) of those in our longitudinal cohort significantly improved after ETI (6.91, SD = 7.74), indicating our study cohort was responsive to ETI. The daily variation of alpha- and beta-diversity of both the microbiome and metabolome was higher for those on ETI, reflecting a more dynamic microbial community and chemical environment during treatment. Four of the seven subjects on ETI were persistently infected with Pseudomonas or Burkholderia in their sputum throughout the sampling period while the total bacterial load significantly decreased with time (R = - 0.42, p = 0.01) in only one subject. The microbiome and metabolome dynamics on ETI were personalized, where some subjects had a progressive change with time on therapy, whereas others had no association with time on treatment. To further classify the augmented variance of the CF microbiome under therapy, we fit the microbiome data to a Hubbell neutral dynamics model in a patient-stratified manner and found that the subjects on ETI had better fit to a neutral model. CONCLUSION: This study shows that the longitudinal microbiology and chemistry in airway secretions from subjects on ETI has become more dynamic and neutral and that after the initial improvement in lung function, many are still persistently infected with CF pathogens.

Decline of a distinct coral reef holobiont community under ocean acidification.

BACKGROUND: Microbes play vital roles across coral reefs both in the environment and inside and upon macrobes (holobionts), where they support critical functions such as nutrition and immune system modulation. These roles highlight the potential ecosystem-level importance of microbes, yet most knowledge of microbial functions on reefs is derived from a small set of holobionts such as corals and sponges. Declining seawater pH - an important global coral reef stressor - can cause ecosystem-level change on coral reefs, providing an opportunity to study the role of microbes at this scale. We use an in situ experimental approach to test the hypothesis that under such ocean acidification (OA), known shifts among macrobe trophic and functional groups may drive a general ecosystem-level response extending across macrobes and microbes, leading to reduced distinctness between the benthic holobiont community microbiome and the environmental microbiome. RESULTS: We test this hypothesis using genetic and chemical data from benthic coral reef community holobionts sampled across a pH gradient from CO2 seeps in Papua New Guinea. We find support for our hypothesis; under OA, the microbiome and metabolome of the benthic holobiont community become less compositionally distinct from the sediment microbiome and metabolome, suggesting that benthic macrobe communities are colonised by environmental microbes to a higher degree under OA conditions. We also find a simplification and homogenisation of the benthic photosynthetic community, and an increased abundance of fleshy macroalgae, consistent with previously observed reef microbialisation. CONCLUSIONS: We demonstrate a novel structural shift in coral reefs involving macrobes and microbes: that the microbiome of the benthic holobiont community becomes less distinct from the sediment microbiome under OA. Our findings suggest that microbialisation and the disruption of macrobe trophic networks are interwoven general responses to environmental stress, pointing towards a universal, undesirable, and measurable form of ecosystem changed. Video Abstract.

Shifts in the functional capacity and metabolite composition of the gut microbiome during recovery from enteric infection.

While enteric pathogens have been widely studied for their roles in causing foodborne infection, their impacts on the gut microbial community have yet to be fully characterized. Previous work has identified notable changes in the gut microbiome related to pathogen invasion, both taxonomically and genetically. Characterization of the metabolic landscape during and after enteric infection, however, has not been explored. Consequently, we investigated the metabolome of paired stools recovered from 60 patients (cases) during and after recovery from enteric bacterial infections (follow-ups). Shotgun metagenomics was applied to predict functional microbial pathways combined with untargeted metametabolomics classified by Liquid Chromatography Mass Spectrometry. Notably, cases had a greater overall metabolic capacity with significantly higher pathway richness and evenness relative to the follow-ups (p<0.05). Metabolic pathways related to central carbon metabolism, amino acid metabolism, and lipid and fatty acid biosynthesis were more highly represented in cases and distinct signatures for menaquinone production were detected. By contrast, the follow-up samples had a more diverse metabolic landscape with enhanced richness of polar metabolites (p<0.0001) and significantly greater richness, evenness, and overall diversity of nonpolar metabolites (p<0.0001). Although many metabolites could not be annotated with existing databases, a marked increase in certain clusters of metabolites was observed in the follow-up samples when compared to the case samples and vice versa. These findings suggest the importance of key metabolites in gut health and recovery and enhance understanding of metabolic fluctuations during enteric infections.

Untargeted metabolomics and metagenomics reveal signatures for intramammary ceftiofur treatment and lactation stage in the cattle hindgut.

The gut microbiota in cattle is essential for protein, energy, and vitamin production and hence, microbiota perturbations can affect cattle performance. This study evaluated the effect of intramammary (IMM) ceftiofur treatment and lactation stage on the functional gut microbiome and metabolome. Forty dairy cows were enrolled at dry-off. Half received IMM ceftiofur and a non-antibiotic teat sealant containing bismuth subnitrate (cases), while the other half received the teat sealant (controls). Fecal samples were collected before treatment at dry off, during the dry period (weeks 1 and 5) and the first week after calving (week 9). Shotgun metagenomic sequencing was applied to predict microbial metabolic pathways whereas untargeted metabolomics was used identify polar and nonpolar metabolites. Compared to controls, long-term changes were observed in the cows given ceftiofur, including a lower abundance of microbial pathways linked to energy production, amino acid biosynthesis, and other vital molecules. The metabolome of treated cows had elevated levels of stachyose, phosphatidylethanolamine diacylglycerol (PE-DAG), and inosine a week after the IMM ceftiofur application, indicating alterations in microbial fermentation, lipid metabolism, energy, and cellular signaling. Differences were also observed by sampling, with cows in late lactation having more diverse metabolic pathways and a unique metabolome containing higher levels of histamine and histamine-producing bacteria. These data illustrate how IMM ceftiofur treatment can alter the functionality of the hindgut metabolome and microbiome. Understanding how antibiotics and lactation stages, which are each characterized by unique diets and physiology, impact the function of resident microbes is critical to define normal gut function in dairy cattle.

Glucocorticoid-induced osteoporosis is prevented by dietary prune in female mice.

Glucocorticoid-induced osteoporosis (GIO) is a significant side effect of prolonged glucocorticoid (GC) treatment. Chronic GC treatment also leads to trabecular bone loss and gut microbiota dysbiosis in mice. The gut dysbiosis is mechanistically linked to GIO, which indicates that the microbiota can be targeted to prevent GIO. Prunes, a dried fruit and prebiotic, have emerged in the literature as an effective treatment for sex-steroid deficiency induced osteoporosis (primary osteoporosis). Prunes also significantly alter the composition of the gut microbiota in both rodent models and human studies. Therefore, we tested if dietary prune (DP) supplementation could prevent GC-induced bone loss and affect microbiota composition in an established model of GIO. Sixteen-week-old, skeletally mature, female C57BL/6J mice were treated with a subcutaneous 5 mg placebo or prednisolone pellet for 8 weeks and fed an AIN-93M control diet or a diet modified to include 5, 15, or 25% (w/w) dried California prune powder. As expected, GC treated mice developed significant trabecular bone loss in the distal femur. More importantly, as little as 5% DP supplementation effectively prevented trabecular bone loss. Further, dose dependent increases in trabecular bone volume fraction were observed in GC + 15% and GC + 25% DP mice. Amazingly, in the placebo (non-GC treated) groups, 25% DP supplementation caused a ∼3-fold increase in distal femur trabecular bone volume fraction; this sizable bone response has not been previously observed in healthy mice with gut targeted natural treatments. Along with the striking effect on bone health, GC treatment and 25% DP supplementation led to drastic shifts in gut microbiota composition and several specific changes are strongly associated with bone health. Taken together, these results are the first to demonstrate that DP supplementation effectively prevents the negative effects of prolonged GC therapy on trabecular bone health and strongly associates with shifts in the composition of the gut microbiota.