Biliary Microbiota In Patients With Calcium Bilirubinate Stone And The Regulation Of L-glutamic Acid And L-arginine On Bacterial β-glucuronidase

Objective: Biliary bilirubinate calcium stone is a clinically intractable disease,and stone formation and recurrence are inextricably related to biliary bacteria.The bacterial information provided by methods such as traditional bacterial culture was limited.With the development of Next Generation Sequencing(NGS),it is essential to gain insight into the composition and function of the biliary microbiota in patients with biliary calcium bilirubinate stones.The origin of biliary tract bacteria has not been clearly elucidated,and since the duodenum and bile duct are anatomically connected and share the same embryonic origin,it is important to study the relationship between the biliary and duodenal microbiota.β-glucuronidase(GUS)produced by Escherichia coli is the key protein responsible for the formation of biliary calcium bilirubinate stone.However,studies focused on bacterial GUS were limited.Various types of bacteria such as E.coli with different serotypes,Klebsiella pneumoniae,and Enterococcus were commonly isolated from the bile duct in patients with biliary calcium bilirubinate stone.Whether these bacteria can express GUS and the differences in enzymatic activity between different bacteria are not known and need to be further explored.Furthermore,from a bacterial perspective,inhibition or reduction of GUS expression by bacteria in the biliary tract was the main breakthrough point for intervention in the formation of calcium bilirubinate stone at present.However,the mechanism of bacterial GUS production and effective interventions have not been fully described.Therefore,exploring the measures and possible regulatory mechanisms that can effectively interfere with the bacterial expression of GUS,will contribute to the treatment and prevention of calcium bilirubinate stone formation.Methods: Bile samples and duodenal juice were collected aseptically from 10 patients who were diagnosed with choledocholithiasis and underwent surgeries at the Second General Surgery Department in Shengjing Hospital of China Medical University.Amplicons of the V3–V4 region of the 16 S r RNA gene were constructed.The composition of the microbiota,Alpha and Beta Diversity,differential microbiota,and the association between microbiota were analyzed.Tax4 Fun was employed for the predictions of the functional profile of a microbial community based on 16 S r RNA sequence data.Then,the common biliary bacteria including E.coli,K.pneumoniae,E.faecalis,E.faecalis,A.baumannii,and P.aeruginosa were compared for GUS expression employing Western blot,quantitative real-time PCR.The reason for bacterial GUS production was also inferred.The composition of free amino acids in the bile from 20 patients was analyzed and compared in patients with primary choledocholithiasis,secondary choledocholithiasis,and control group(adenomatous polyps of gallbladder or adenomyosis of gallbladder)using targeted metabolomics.The effects of complementary differential amino acids on bacterial GUS expression were examined at the level of GUS enzyme activity,GUS protein,and mRNA level.Finally,the role of the uidR gene in the regulation of bacterial GUS expression by differential amino acids was investigated by constructing the uidR gene knockout strain of E.coli.Results: The dominant phyla for both the biliary and duodenal microbiota were Proteobacteria and Firmicutes.The core microbiota analyzed in the Microbiome Analyst platform was similar between biliary and duodenal samples at the genus level,with the top three being Escherichia–Shigella,Fusobacterium,and Enterococcus.Of the annotated species in the phylum Proteobacteria,E.coli accounted for the most abundant in both biliary and duodenal microbial communities.Alpha-diversities revealed no significant differences in enrichment and evenness between biliary and duodenal microbiota(ACE: P= 0.579;Shannon: P = 0.089,by Wilcoxon rank-sum test).Principal coordinates analysis showed no distinct clustering in the biliary and duodenal microbiota,and no significant difference in microbial composition was observed(Amova: P = 0.509).A LEf Se algorithm indicated the genus Streptococcus and an unidentified genus of Actinobacteria as the microbial features characterizing duodenal juice from bile in patients with choledocholithiasis;however,no taxa were determined by LEf Se to be characteristic of the biliary microbiota compared with the duodenal microbiota.Functional profiles were obtained through Tax4 Fun based on a KEGG database.The major identified pathways of biliary and duodenal microbiota were related to membrane transport,translation,replication and repair,carbohydrate metabolism,and amino acid metabolism.Like microbial composition,differences in major predicted functional profiles between biliary and duodenal microbiota were not significant.Subordinate pathways of carbohydrate metabolism and amino acid metabolism were also analyzed and compared.In both groups of biliary and duodenal microbiota,pyruvate metabolism,glycolysis/gluconeogenesis,amino sugar,and nucleotide sugar metabolism were enriched in carbohydrate metabolism,while alanine,aspartate,and glutamate metabolism were enriched in amino acid metabolism(relative abundance > 1%).However,no significant distinction was observed between biliary and duodenal microbiota.Meanwhile,based on the KEGG database and functional analysis,it was found that the main metabolic pathway involved in the formation of calcium bilirubinate stone by bacterial GUS was the D-glucuronide degradation pathway,whose products continued into the pentose phosphate pathway and eventually produce pyruvate.Common bacteria from patients with calcium bilirubinate stone such as E.coli(serotypes O24,O30,O79),K.pneumoniae,E.faecalis,E.faecalis,A.baumannii,and P.aeruginosa were selected for GUS enzyme activity assay,and it was found that no enzymatic reaction occurred in all strains after incubation in nutrient broth;while when the above bacteria were incubated with bile,the three serotypes of E.coli,E.faecium,and E.faecalis showed enzymatic reactions,while K.pneumoniae,A.baumannii,and P.aeruginosa did not show enzymatic reactions.The supernatant(bile)and precipitate(bacteria)of bile incubated with E.coli and E.faecium were assayed for GUS enzyme activity separately,and it was found that the enzymatic reaction occurred only in the fraction with bacteria,while no enzymatic reaction occurred in the fraction the bile.Comparison of the GUS enzyme activity assays for the above bacteria with GUS enzyme activity revealed that the order of strength of GUS enzyme activity was: E.coli O30 > E.coli O79 > E.faecium > E.coli O24 > E.faecalis.The expression of GUS protein and uid A mRNA of E.coli was compared between nutrient broth and bile incubation,and it was found that bile incubation significantly up-regulated the expression of GUS protein and mRNA compared to the nutrient broth.GUS protein expression was also significantly higher in E.coli O30 and O79 than in E.coli O24,and the difference was statistically significant.Similarly,the differences in uid A mRNA expression among E.coli O24,O30,and O79 were also statistically significant.A possible intervening factor for bacterial GUS biliary amino acids was inferred.The bile samples from different groups were tested for targeted amino acid and analyzed,and the differential amino acids were found to be L-glutamic acid,L-arginine,and creatine,with all three being significantly lower in patients with primary choledocholithiasis.It was found that L-glutamic acid and L-arginine,except for creatine,inhibited bile-induced GUS enzyme activity in E.coli O30 and E.faecium in a concentration-dependent manner.Lglutamic acid and L-arginine down-regulated E.coli O30 and O79 GUS protein and mRNA expression.Furthermore,the intervention of GUS enzymes extracted from E.coli with Lglutamic acid revealed no statistically significant difference in the slope S of the enzyme reaction trend line,which represents the enzyme reaction rate(i.e.,enzyme activity),compared with the group without the addition of L-glutamic acid at the same GUS enzyme concentration.The uidR mRNA expression level of E.coli O30 was significantly lower after incubation with bile compared to nutrient broth culture conditions,and was significantly higher than the pre-intervention level after the administration of L-glutamic acid(150 μg/ml)and Larginine(500 μg/ml)interventions.E.coli with the GUS repressor gene uidR knocked out also expressed GUS protein even cultured in nutrient broth,and similar results were observed at the mRNA level assay.Knockdown of uidR significantly reversed the inhibitory effects of L-glutamic acid and L-arginine on the expression of GUS protein and mRNA.Conclusion: Biliary microbiota shared a similar composition and function with duodenal microbiota in patients with calcium bilirubinate stone.Bacterial GUS expression was conditioned and bile was its inducing factor.Furthermore,bacterial GUS was expressed inside bacterial cells rather than being secreted into the extracellular environment.Among the common biliary tract bacteria in patients with biliary calcium cholestasis,E.coli(serotypes O24,O30,O79),E.faecium,and E.faecalis were able to express GUS,and their enzymatic activity varied.After incubation with bile,there were also statistical differences in protein and mRNA expression levels of GUS in different serotypes of E.coli O24,O30,and O79.It was inferred that the mechanism of GUS production by biliary bacteria from patients with calcium bilirubinate stone was to take up glucuronides from conjugated bilirubin as a carbon source for energy and metabolism,reflecting the scarcity of carbon sources for bacteria in bile.Bile was also relatively depleted in L-glutamic acid,L-arginine,and creatine,and supplementation with L-glutamic acid and L-arginine inhibited bile-induced GUS enzyme activity in E.coli O30 and E.faecium,and reduced bile-induced GUS expression in E.coli O30 and O79 at the protein and mRNA levels,and this inhibition was not a direct effect of L-glutamic acid.The GUS repressor gene uidR played a major regulatory role in the inhibition of GUS expression by L-glutamic acid and L-arginine.