Identifying a Novel Bile Salt Hydrolase from the Keystone Gut Bacterium Christensenella minuta

Christensenella minuta are human gut dwelling bacteria that have been proposed as key members of the gut microbiome, regulating energy balance and adiposity of their host. We formerly identified that a novel strain of C. minuta (strain DSM33407) boosted microbiota diversity and stimulated deconjugation of the primary bile acid taurocholic acid in human samples. However, there is no description of a bile salt hydrolase (BSH) protein carried in the genome of C. minuta. Here, we identified and cloned a protein from C. minuta’s genome that carries a potent BSH activity, which preferentially deconjugates glycine-conjugated bile acids. We then retrieved 14,319 putative BSH sequences from the NCBI database and filtered them using the UHGP database to collect a total of 6701 sequences that were used to build the most comprehensive phylogenetic tree of BSH-related enzymes identified in the human microbiome so far. This phylogenetic tree revealed that C. minuta’s BSH amino acid sequence clusters away from others with a threshold of 70% identity. This is therefore the first description of C. minuta’s BSH protein, which may be involved in its unique role within the human gut microbial ecosystem.

Compact Miniaturized Bioluminescence Sensor Based on Continuous Air-Segmented Flow for Real-Time Monitoring: Application to Bile Salt Hydrolase (BSH) Activity and ATP Detection in Biological Fluids

A simple and versatile continuous air-segmented flow sensor using immobilized luciferase was designed as a general miniaturized platform based on sensitive biochemiluminescence detection. The device uses miniaturized microperistaltic pumps to deliver flows and compact sensitive light imaging detectors based on BI-CMOS (smartphone camera) or CCD technology. The low-cost components and power supply make it suitable as out-lab device at point of need to monitor kinetic-related processes or ex vivo dynamic events. A nylon6 flat spiral carrying immobilized luciferase was placed in front of the detector in lensless mode using a fiber optic tapered faceplate. ATP was measured in samples collected by microdialysis from rat brain with detecting levels as low as 0.4 fmoles. The same immobilized luciferase was also used for the evaluation of bile salt hydrolase (BSH) activity in intestinal microbiota. An aminoluciferin was conjugatated with chenodeoxycholic acid forming the amide derivative aLuc-CDCA. The hydrolysis of the aLuc-CDCA probe by BSH releases free uncaged aminoluciferin which is the active substrate for luciferase leading to light emission. This method can detect as low as 0.5 mM of aLuc-CDCA, so it can be used on real faecal human samples to study BSH activity and its modulation by diseases and drugs.

Identification of Bile Salt Hydrolase and Bile Salt Resistance in a Probiotic Bacterium Lactobacillus gasseri JCM1131T

Lactobacillus gasseri is one of the most likely probiotic candidates among many Lactobacillus species. Although bile salt resistance has been defined as an important criterion for selection of probiotic candidates since it allows probiotic bacteria to survive in the gut, both its capability and its related enzyme, bile salt hydrolase (BSH), in L. gasseri is still largely unknown. Here, we report that the well-known probiotic bacterium L. gasseri JCM1131T possesses BSH activity and bile salt resistance capability. Indeed, this strain apparently showed BSH activity on the plate assay and highly tolerated the primary bile salts and even taurine-conjugated secondary bile salt. We further isolated a putative BSH enzyme (LagBSH) from strain JCM1131T and characterized the enzymatic function. The purified LagBSH protein exhibited quite high deconjugation activity for taurocholic acid and taurochenodeoxycholic acid. The lagBSH gene was constitutively expressed in strain JCM1131T, suggesting that LagBSH likely contributes to bile salt resistance of the strain and may be associated with survival capability of strain JCM1131T within the human intestine by bile detoxification. Thus, this study first demonstrated the bile salt resistance and its responsible enzyme (BSH) activity in strain JCM1131T, which further supports the importance of the typical lactic acid bacterium as probiotics.

Selection of potential probiotics with cholesterol-lowering properties for probiotic yoghurt production

From 61 lactic acid bacteria (LAB) isolates, three had good cholesterol-lowering properties, with Limosilactobacillus fermentum KUB-D18 having the highest cholesterol assimilation (68.75%) (51 µg/109 CFU). In addition, Lactiplantibacillus pentosus HM04-25 and L. pentosus HM04-3 had the two highest levels of bile salt hydrolase (BSH) activity (22.60 and 21.45 U/mL, respectively). These three strains could resist four antibiotics (aztreonam, vancomycin, teicoplanin, and nalidixic). However, fortunately, they contained no mobile antibiotic resistance genes. To evaluate the influence of probiotic strains in yoghurt production, L. fermentum KUB-D18, L. pentosus HM04-25, or L. pentosus HM04-3 were simultaneously cultured with commercial yoghurt starter (YF-L812) and incubated at 43 °C for 6 h. During yoghurt fermentation, the total bacteria in the yoghurt tended to increase from 7.39 to 8.90 log CFU/mL. The growth rates of two probiotic strains ( L. pentosus HM04-25 and L. pentosus HM04-3) were stable at 6.06 to 6.62 log CFU/mL. Only the rate for L. fermentum KUB-D18 increased (to 7.5 log CFU/mL). These three probiotics did not affect the physical characteristics of yoghurt. The total soluble solids, pH, and titratable acidity values of the probiotic yoghurts were similar to the control yoghurt at 30[Formula: see text]Brix, 4.91, and 0.90%, respectively. The firmness values of the probiotic yoghurts and the control were not significantly different (p > 0.05). Differentiation of the appearance of color, odor, flavor, and texture between the control yoghurt and the probiotic yoghurts was investigated using 56 volunteers and no significant differences were identified. Additionally, sensory evolution revealed that the acceptability of the probiotic yoghurts was higher than for the control (p ≤ 0.05). Therefore, the three probiotic strains with cholesterol-lowering properties had potential in future yoghurt production.

Bile salt hydrolases deplete conjugated bile acids and erode gut barrier integrity in non-alcoholic steatohepatitis

Background & Aims: While altered host-microbe interactions are implicated in non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), specific contributions of microbially derived metabolites remain obscure. We investigated the impact of altered bile acid (BA) populations on intestinal and hepatic phenotypes in a rodent model of NAFLD/NASH. Methods: Wistar rats fed a choline-deficient high-fat diet (CDAHFD) were assessed for altered intestinal permeability after dietary intervention. Cecal and portal venous BA composition were assessed via mass spectrometry. BA-mediated effects on epithelial permeability were assessed using Caco2 epithelial monolayers. Micelle formation was assessed using fluorescent probes and electron microscopy. Bile salt hydrolase (BSH) activity was inhibited with a gut-restricted small molecule in CDAHFD-fed rats and intestinal and hepatic phenotypes were assessed. Results: Increased intestinal permeability and reduced intestinal conjugated BAs were early phenotypes of CDAHFD-fed rats preceding hepatic disease development. Similar intestinal BA pool changes were observed in rats and human NAFLD/NASH patients with progressive disease. Conjugated BAs protected epithelial layers from unconjugated BA-induced damage via mixed micelle formation. The decrease in intestinal conjugated BAs was mediated by increased activity of bacterial BSHs and inhibition of BSH activity prevented the development of pathologic intestinal permeability and hepatic inflammation in the NAFLD/NASH model. Conclusions: Conjugated BAs are important for the maintenance of intestinal barrier function by sequestering unconjugated BAs in mixed micelles. Increased BSH activity reduces intestinal conjugated BA abundance, in turn increasing intestinal permeability and susceptibility to the development of NAFLD/NASH. These findings suggest that interventions that shift the intestinal bile acid pool toward conjugated BAs could be developed as therapies for NAFLD/NASH.

In Vitro Bile Salt Hydrolase (BSH) Activity Screening of Different Probiotic Microorganisms

Bile salt hydrolase (BSH) activity in probiotic strains is usually correlated with the ability to lower serum cholesterol levels in hypercholesterolemic patients. The objective of this study was the evaluation of BSH in five probiotic strains of lactic acid bacteria (LAB) and a probiotic yeast. The activity was assessed using a qualitative direct plate test and a quantitative high-performance thin- layer chromatography assay. The six strains differed in their BSH substrate preference and activity. Lactobacillus plantarum DGIA1, a potentially probiotic strain isolated from a double cream cheese from Chiapas, Mexico, showed excellent deconjugation activities in the four tested bile acids (69, 100, 81, and 92% for sodium glycocholate, glycodeoxycholate, taurocholate, and taurodeoxycholate, respectively). In the case of the commercial probiotic yeast Saccharomyces boulardii, the deconjugation activities were good against sodium glycodeoxycholate, taurocholate, and taurodeoxycholate (100, 57, and 63%, respectively). These last two results are part of the novelty of the work. A weak deconjugative activity (5%) was observed in the case of sodium glycocholate. This is the first time that the BSH activity has been detected in this yeast.

A gut-restricted lithocholic acid analog as an inhibitor of gut bacterial bile salt hydrolases

Bile acids play crucial roles in host physiology by acting as both detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. SAR studies focused on the bile acid core identified a compound, AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in conventional mouse fecal slurries, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration of AAA-10 to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure of AAA-10, demonstrating that AAA-10 is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo.

Noninvasive imaging and quantification of bile salt hydrolase activity: From bacteria to humans

The microbiome-produced enzyme bile salt hydrolase (BSH) plays a central role in human health, but its function remains unclear due to the lack of suitable methods for measuring its activity. Here, we have developed a novel optical tool based on ultrasensitive bioluminescent imaging and demonstrated that this assay can be used for quick and cost-effective quantification of BSH activity across a broad range of biological settings including pure enzymes and bacteria, intact fecal slurries, and noninvasive imaging in live animals, as well as for the assessment of BSH activity in the entire gastrointestinal tract of mice and humans. Using this assay, we showed that certain types of prebiotics are capable of increasing BSH activity of the gut microbiota in vivo and successfully demonstrated potential application of this assay as a noninvasive diagnostic test to predict the clinical status of inflammatory bowel disease (IBD) patients.

MODIFICATION OF GUT BACTERIAL BILE SALT HYDROLASE ACTIVITY AND CARDIOVASCULAR RISK: A RANDOMIZED STUDY

Introduction: secretion of bacterial bile salt hydrolase (BSH) is one of the main mechanisms by which gut microbiota play role in cholesterol metabolism. There are limiting and controversial data regarding the clear effect of gut BSH activity correction on modification of serum cholesterol and cardiovascular risk (CVR). Aim of investigation was to evaluate the relationship between modification of the gut bacterial BSH relative activity (RA) by probiotic L. plantarum and serum cholesterol with CVR levels. Methods: the study was conducted as open, comparative, randomized, parallel and included 26 almost healthy participants (healthy control group) and 77 patients with dyslipidemia and without anamnesis of major cardiovascular events, that were divided in two groups: main treatment group (n=41) received combination therapy (capsules with Lactobacillus plantarum in the amount of 2*109 CFU one time a day and tablets simvastatin 20 mg one time a day) and control treatment group (n=36) received monotherapy (simvastatin 20 mg one time a day) during 12 weeks. Before and after 12 weeks of treatment the assessment of total RA of gut BSH, lipid profile and CVR level according to 5 risk scores were performed. Results: at baseline the RA of BSH was higher in healthy adults comparing to participants with dyslipidemia (p<0,001); after 12 weeks of treatment there wasn`t difference between healthy control and only main treatment groups (р=0,45). It was found that with increasing of RA of gut bacterial BSH, the risk of failure of treatment efficacy endpoints achievement (³20% reduction of values) decreased regarding: total cholesterol (TC) (p=0,0306), OR=0,00133 (95% CI; 3,28*10-6-0,538); low-density lipoproteins (LDL) (p<0,001), OR=5,65*10-14(95% CI; 6,38*10-20-5*10-8); CVR level according to Framingham score (р=0,0035), OR=4,09*10-5(95% CI; 4,66*10-8-0,0359); CVR level according to 2013 ACC/AHA algorithm (р=0,0135), OR=3,8*10-4(95% CI; 7,34*10-7-0,197); CVR level according to PROCAM score (p=0,00125), OR=8,38*10-6 (95%; CI; 6,93*10-9-0,0101). Conclusions: additional supplementation with BSH-producing bacteria L. plantarum was more effective in increasing of BSH activity compared to simvastatin monotherapy. Increasing of BSH RA by L. plantarum was associated with higher chances to achieve treatment efficacy goals regarding reduction of TC, LDL and CVR levels according to Framingham, 2013 ACC/AHA algorithm and PROCAM scores.

Probiotic Potential of Lactic Acid Starter Cultures Isolated from a Traditional Fermented Sorghum-Millet Beverage

The purpose of this study was to establish the probiotic potential of lactic acid bacteria (LAB) starter cultures, Lb. plantarum MNC 21, L. lactis MNC 24, and W. confusa MNC 20, isolated from a traditionally fermented sorghum-millet beverage from Uganda. The cultures were examined for tolerance to acid and bile salts, bile salt hydrolase (BSH) activity, antibiotic susceptibility, biogenic amine production, mucin degradation, hydrophobicity, auto-aggregation, adherence to the ileum, coaggregation, and antimicrobial properties against selected pathogenic species. Lb. rhamnosus yoba 2012, a known probiotic, was the reference. The isolates were tolerant to acid (pH = 3) and bile (1%). W. confusa MNC 20 and Lb. plantarum MNC 21 exhibited medium BSH activity (11–15 mm diameter of hydrolysis zone) while L. lactis and Lb. rhamnosus yoba 2012 exhibited low BSH activity (<10 mm diameter of hydrolysis zone). All isolates lacked mucolytic activity. Lb. plantarum MNC 21 and W. confusa MNC 20 produced agmatine. The candidate and reference microorganisms were resistant to 10 of 21 and 5 of 21 antibiotics, respectively. The isolates exhibited hydrophobic, auto-aggregation and coaggregation properties. These three properties were exhibited more (p<0.05) by the reference than the potential probiotics. The ability of the potential probiotics to attach onto the goat ileum (7.3–8.0 log cfu/cm2) was comparable to that of Lb. rhamnosus yoba 2012 (7.6 log cfu/cm2). The four LAB inhibited E. coli, S. aureus, and S. enterica to the same extent (p<0.05). The findings indicated potential probiotic activity of the starter cultures. However, further in vivo examination of these isolates is required to confirm their probiotic capabilities.