Escherichia coli expressing the kpsM gene exacerbates drug-induced liver injury through up-regulating α1,2-fucosyltransferase and disturbing the host taurine metabolism—from animal models and clinical studies

Graphical abstract

Highlights

• •The abundance of E. coli was the highest and positively associated with the indicators of liver damage in DILI patients.
• •The detection rate of kpsM⁺ E. coli was higher in moderate-to-severe DILI patients compared to mild DILI patients.
• •The kpsM⁺ E. coli aggravated the APAP-induced liver injury in wide type but not intestinal epithelial Fut2 knockout mice.
• •Fut2 inhibited the intestinal transport of taurine, and decreased taurine and its downstream product TUDCA in the liver.
• •The plasma level of taurine was reduced significantly in moderate-to-severe DILI compared to mild DILI.

Abstract

Background and aims

Drug-induced liver injury (DILI) is a leading cause of acute liver failure. Patients with DILI have disorders of the gut microbiota, yet little is known about the influence of gut microbes on this disease. Herein, we investigated the alterations of gut microbiota in DILI patients, and elucidated the mechanism by which Escherichia coli expressing kpsM gene (kpsM⁺E. coli) exacerbates DILI, in order to provide targets for intervention of related signaling pathways to improve DILI.

Methods

Full-length 16S sequencing was performed on fecal samples from a prospective cohort of patients with DILI (n = 42). Quantitative PCR was employed for analysis of E. coli and its kpsM gene in human feces. The DILI model was established by intraperitoneal injection of acetaminophen (300 mg/kg) into mice (n = 5–12). Two hours later, kpsM⁺ or kpsM knockout E. coli strains were gavaged to determine their roles during DILI. Intestinal epithelial Fut2 gene knockout mice (Fut2^ΔIEC) and hepatic metabolome were used to assess the pathogenic mechanisms of the kpsM⁺E. coli. Plasma metabolome of DILI patients was further validated the discoveries in mice.

Results

The percentage of subjects carrying kpsM were 14.7 %, 40.0 %, 76.5 % in healthy controls, patients with mild DILI, and patients with moderate-to-severe DILI, respectively. Mice transplanted with kpsM⁺E. coli exhibited more severe DILI, primarily achieved through impaired gut barrier function and enhanced expression of intestinal Fut2. Fut2^ΔIEC mice alleviated the aggravation of DILI caused by E. coli via up-regulating the hepatic levels of taurine and tauroursodeoxycholic acid. In addition, the level of plasma taurine was lower in patients with moderate-to-severe DILI than in those with mild DILI.

Conclusions

The kpsM⁺ E. coli was associated with the severity of DILI in human. This strain can exacerbate DILI in mice through up-regulating intestinal Fut2 expression and disrupting taurine metabolism.

Introduction

With the aging of the population, the increasing prevalence of chronic diseases, and the widespread use of novel biologics, the global incidence of drug-induced liver injury (DILI) has been rising year by year [1]. DILI is also the leading cause of acute liver failure, responsible for more than 50 % of cases [2]. There are geographical differences in the types of medications that cause this disease, with anti-tuberculosis drugs and traditional Chinese medicine dominating in Asia, while acetaminophen (APAP)-induced liver injury (AILI) is most frequent in the West [3].

Recent research suggests an essential role of the gut microbiota in the development of DILI. Different types of hepatotoxic drugs can lead to abnormalities in the gut microbiota of the host [4,5]. Several clinical studies have found disturbances in the gut microbiota of patients with DILI [6,7], and supplementation of beneficial bacteria like Lactobacillus and Akkermansia muciniphila can improve DILI [[8], [9], [10], [11], [12], [13]]. Although many efforts were made to assess the role of probiotics in DILI, little is known about the mechanisms of specific pathobionts and their virulence factors in this disease. Especially, the interaction between bacteria and their host, and the contribution of the different genotypes of the bacterial species to DILI remains poorly understood.

Escherichia coli (E. coli) is a bacterium widely present in nature and the human intestine, which maintains intestinal health and function. However, some strains, such as enteropathogenic or enterohemorrhagic E. coli, are pathogenic, which can damage gut barrier, cause diarrhea, infections, and liver diseases. For example, adherent-invasive E. coli increases epithelial permeability through disrupting mitochondrial function [14]. E. coli exacerbates non-alcoholic fatty liver disease by promoting mesenchymal transformation in liver sinusoidal endothelial cells [15] and disrupting the vascular barrier [16]. Previous research indicates that the kpsM gene is primarily present in E. coli, encoding a capsule polysaccharide (CPS) transporter protein that is responsible for transporting CPS to the bacterial cell surface [17]. CPS usually functions as a key virulence factor for E. coli [18], which facilitates bacterial colonization and impedes its removal by the host immune system [19,20]. Deletion of the kpsM leads to a weaker resistance of E. coli to serum killing, and reduces its adhesion and anti-phagocytic capabilities, thereby diminishing its virulence [21]. Whether kpsM⁺ E. coli interacts with intestinal epithelial cells or affects gut barrier function needs further investigation.

The key pathological feature of DILI is the disruption of bile acid homeostasis, and harmful bile acids also exacerbate the progression of DILI [22]. Of note, bile acids and gut microbiota exhibit bidirectional interactions. Hepatotoxic drugs alter gut microbiota, which influences bile acid metabolism, while bile acids also affect the composition and structure of intestinal microbes [23]. For example, depleting gut microbiota with antibiotics reduced taurine concentration in mice [24], and supplementation of exogenous taurine altered the microbiota function, providing it with anti-infective capabilities [25]. However, the specific bacterial strains that influence bile acid metabolism have not yet been fully studied. In human patients, the relative abundance of E. coli showed a significant positive correlation with serum total bile acid levels [21]. Animal experiments confirm that E. coli infection can disrupt bile acid homeostasis [26]. Given that kpsM⁺ E. coli exhibits enhanced colonization capacity, whether and how it perturbs bile acid profiles in DILI awaits further study.

Alpha1,2-fucosyltransferase (Fut2) is an enzyme that transfers L-fucose to N-acetylglucosamine and mannose residues, forming specific glycan structures. These structures function critically in immune responses, intercellular adhesion, and signal transduction. Our previous research indicates that Fut2 deficiency significantly upregulates the bacterial enzymes 7-α-hydroxysteroid dehydrogenases, a bacterial enzyme involved in bile acid metabolism, and attenuates Western diet-induced dysregulation of bile acid metabolism [27]. Notably, functional variations in Fut2 also affect the composition and structure of the host's gut microbiota [28] and the intestinal immunity [29]. In patients with the loss-of-function mutation of Fut2 gene, the only bacterial genus showing a significant decrease in fecal samples is Escherichia [28]. The Fut2-deficient mice exhibit significant alterations in the Escherichia [30]. E. coli stimulation promotes the expression of the Fut2, while knockdown of Fut2 reduces E. coli adhesion to intestinal epithelial cells [31].

In summary, the above evidence indicates a potential interaction between E. coli, the intestinal Fut2 expression, and the host bile acid metabolism. In this study, we prospectively collected fecal specimens from patients with DILI of different severity, and performed full-length 16S bacterial sequencing, which found that the abundance of E. coli was positively associated with the development of DILI. Through bacterial transplantation, we explored the impacts of different genotypes of E. coli on mice with AILI, and identified Fut2 as a key host gene involved in the pathogenic process of E. coli. Transgenic mice further clarified that Fut2-deficient influenced the metabolite profiles, providing a new perspective for the clinical prevention and treatment of DILI.

Method and materials

Patient selection and clinical trial design

A total of 42 patients diagnosed with DILI were prospectively recruited from Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. All the DILI patients included in this study suffered from acute liver damage, with the primary causative agents being traditional Chinese medicines or herbal remedies. They met the following inclusion criteria: (1) age over 18 years; (2) clinically diagnosed with acute DILI; (3) history of taking medications known to cause liver injury; (4) no administration of antibiotics or probiotics in the month prior to enrollment; (5) relatively complete clinical data, agreement to participate in this clinical study, and signed informed consent forms. The exclusion criteria: (1) Patients using amoxicillin-clavulanate potassium, antiepileptics (such as sodium valproate), antituberculosis drugs (such as isoniazid, rifampicin), antitumor drugs (such as methotrexate, cyclophosphamide), and statins; (2) patients with hepatocellular carcinoma or hepatic metastasis; (3) coexistence of viral liver diseases, including non-hepatotropic viruses such as cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus-related hepatitis, and hepatotropic virus-induced hepatitis A, B, C, D, and E; (4) coexistence of the non-infectious liver diseases, like non-alcoholic fatty liver disease, IgG4-related hepatitis, hepatolenticular degeneration, α1-antitrypsin deficiency, hemochromatosis, Budd-Chiari syndrome, congenital and inherited liver diseases; (5) coexistence of severe pathology in other organs, such as renal failure, heart failure, etc.; (6) pregnant or lactating women. The inclusion criteria for the HC group: (1) age over 18 years; (2) no history of liver disease in the past year; (3) no other acute or chronic medical histories in the past year; (4) no history of taking antibiotics or probiotics in the past month; (5) agreement to participate in this clinical study, and signed informed consent forms.

Based on Chinese clinical practice guidelines and previous reports [32,33], the classification of severity of liver injury in DILI patients was as follows: Grade 1, mild liver damage: total bilirubin (TB) < 2.5 times the Upper Limit of Normal (ULN) (42.75 μmol/L in this study) and the International Normalized Ratio (INR) is less than 1.5. Grade 2, moderate liver damage: TB ≥ 2.5 times ULN, or INR ≥ 1.5 although there is no increase in TB. Grade 3, severe liver damage: TB ≥ 5 times ULN (85.5 μmol/L in this study), with or without INR ≥ 1.5. Grade 4, acute liver failure: TB ≥ 10 times ULN (171.0 μmol/L) or a daily rise of 17.1 μmol/L), INR ≥ 2.0 or prothrombin activity <40 %, may be accompanied by ascites or hepatic encephalopathy, or other organ failure associated with DILI. Patients meeting the criteria of Grade 2 to Grade 4 were collectively referred to as the Moderate-to-Severe group, while the Grade 1 patients were classified as the Mild group. The clinical data of the subjects were presented in Table S1.

For fecal sample collection, patients were instructed to avoid contamination of the samples with urine, water, or other substances. An appropriate amount of mid-section fecal sample was placed into a 5 mL sterile tube and stored long-term at −80 °C. For plasma sample collection, more than 3 mL of blood was drawn using a heparin anticoagulant tube, centrifuged at 3000 rpm for 15 min at 4 °C, and then an appropriate amount of supernatant was aliquoted into a 2 mL sterile cryopreservation tube and stored long-term at −80 °C.

The research protocol was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, and registered on ClinicalTrials (https://clinicaltrials.gov, NCT05465642). All subjects participating in this project signed informed consent forms.

Full-length 16S rRNA sequencing

The detailed procedure could be found in our previously published articles [34]. In brief, 16S rRNA gene of bacteria were amplified using universal bacterial primers 27F (5′-AGRGTTYGATYMTGGCTCAG-3′) and 1492R (5′-RGYTACCTTGTTACGACTT-3′) [35]. Use USEARCH11-uparse (https://www.drive5.com/uparse, version 11) to perform OTU clustering algorithm on the data based on 97 % similarity. Taxonomic annotation of the OTUs was performed using the RDP classifier (https://rdp.cme.msu.edu, version 11.5) against the NT_Taxon_core_v2024/16s_bacteria database with a confidence threshold of 70 %. The α-diversity indices were measuring using the Shannon and Simpson indices. Principal Coordinates Analysis (PCoA) algorithms were employed to analyze β-diversity among groups. Redundancy analysis (RDA) was used to visualize the bacterial structure and determine its correlation with clinical parameters.

Fecal qPCR for kpsM

Fecal genomic DNA was extracted using a Fecal Genomic DNA Extraction Kit (Tiangen, China) based on the manufacturer’s instructions. The concentration and purity of extracted DNA were measured using a NanoDrop 2000. Qualified DNA samples were used for subsequent qPCR. The qPCR reaction mixture included 10 μL of SYBR green master mix (Vazyme, Nanjing, China), 1 μL of a 10 μM primer mix (forward and reverse), and 9 μL of DNA at a concentration of 10 ng/μL. A cut-off cycle threshold value of <30 was regarded as the presence of kpsM. The nucleotide sequence of the kpsM gene was showed in Table S4, the complete genome used in this study can be found in Genbank: CP006632.1 (https://www.ncbi.nlm.nih.gov/nuccore/CP006632.1/).

Cultivation of E. coli

The E. coli strain was a generous gift from the Laboratory of College of Veterinary Medicine, Huazhong Agricultural University. The kpsM-knockout E. coli was validated and published in previous literature [36]. They were proliferated in Luria-Bertani (LB) solid medium or liquid medium for subsequent gavage and experiments.

In vitro phagocytosis assay of Kupffer cells

The immortalized mouse hepatic Kupffer cells were purchased from Xinrun Biotechnology Co., Ltd (Wuxi, China). They were seeded into 24-well plates (10⁵ cells per well), followed by co-culture with E. coli at a 10:1 ratio for 2 h at 37 °C. Wash with PBS, then add DMEM medium containing chloramphenicol to each well. After 1 h, wash cells with phosphate buffered saline (PBS) and lyse them with sterile deionized water. Collect the lysate, perform serial dilutions, and plate onto LB solid agar to determine the number of surviving bacteria. The average adherence rate of the kpsM⁺ E. coli was set as 100 %.

In vitro adhesion assay on Caco2 cells

The Caco2 cells were purchased from Zishan Biotechnology Co., Ltd (Wuhan, China). Seed Caco-2 cells into a 24-well plate (10⁶ cells per well). Subsequently, add E. coli at a 10:1 ratio for co-culture with the cells. Incubate at 37 °C for 4 h, then wash five times with PBS. Lysate the cells using deionized water. Perform serial dilutions of the lysate and plate onto LB agar plates to enumerate adherent viable bacteria. The average survival rate of the kpsM⁺ E. coli was set as 100 %.

Animals and treatment

The male C57BL/6J mice (aged 7–8 weeks with a body weight range of 22–24 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The intestinal epithelial cell-specific Fut2 gene knockout mice (Fut2^ΔIEC) were constructed using Pvillin-Cre recombinase transgenic C57BL/6J mice (Pvillin-Cre TG mice) and Fut2^flox/flox C57BL/6J mice, which were fed in Shulaibao Biotechnology Co., Ltd. and were verified in our previous study [30].

An AILI mouse model was constructed by intraperitoneal injection of APAP (300 mg/kg body weight, dissolved in PBS before use), with the control group receiving intraperitoneal injection of PBS. To explore the effect of E. coli on AILI, mice were gavaged with E. coli before or 2-hour after APAP administration. Two hours post-injection, the mice were gavaged with sterile PBS, kpsM-knockout E. coli, or kpsM-positive E. coli (5 × 10¹⁰ CFU/kg body weight of the mouse), respectively. To explore the role of host Fut2 gene on AILI, Fut2^ΔIEC and Fut2^fl/fl mice were injected with APAP (300 mg/kg body weight) and gavaged with sterile PBS, kpsM-knockout E. coli, or kpsM-positive E. coli (5 × 10¹⁰ CFU/kg body weight of the mouse) two hours later. To explore the therapeutic effects of taurine, mice were gavaged with taurine (300 mg/kg body weight, dissolved in PBS before use) before APAP injection. All the hepatic and intestinal samples, and blood from the inferior vena cava were collected 24 h after APAP injection.

To identify the influence of intestinal Fut2 gene on the level of taurine, Fut2^ΔIEC and Fut2^fl/fl mice were gavaged with a high dose of taurine (300 mg/kg body weight, dissolved in PBS before use). Cecum content, liver, and blood were collected after 3 h gavage. According to previous reports, the total gastrointestinal transit time (i.e., the time required for food to be ingested and excreted as feces) in male C57 mice ranges from 97 to 162 min at room temperature [37]. Thus, the taurine levels were detected after 3 h to ensure its adequate absorption by the intestine.

Fecal microbiota transplantation and depletion experiment

Collect human feces and resuspend in PBS (0.125 g/mL), then centrifuge briefly and collect the supernatant for subsequent use. Mice were administered antibiotics via gavage for 5 days (200 mg/kg neomycin sulfate, 200 mg/kg metronidazole, 200 mg/kg ampicillin, and 100 mg/kg vancomycin), followed by transplantation of fecal suspensions from DILI patients or healthy individuals for 3 days. Recipient mice then received intraperitoneal injections of APAP (300 mg/kg) to observe the effects of human feces on the development of DILI.

Biochemical analysis

The blood of the mice was centrifuged at 3000 rpm for 15 min to obtain the supernatant plasma. ALT and AST kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) were used to detected the liver enzyme activities. All operations were carried out according to the test instructions.

Histopathological analysis

Liver tissues were stained with hematoxylin eosin to observe hepatic necrosis and inflammation.

Ulex europaeus agglutinin-I (UEA-I) staining

The frozen slides were incubated with rhodamine UEA-I for 1 h at 37 °C. DAPI was used to stain the nuclei. Finally, images were acquired with a fluorescence microscope (Olympus, Japan). Fluorescence intensities were analyzed and semi-quantified using ImageJ software.

Animal tissue RNA extraction, reverse transcription, and real-time quantitative PCR

RNA was isolated from mice liver using RNA easy isolation kit (Vazyme, Nanjing, China) and reversed transcribed to cDNA. Gene expression was determined with SYBR Green (Vazyme, Nanjing, China) using Roche Applied Science LightCycler 480 system. Primer sequences were obtained from PrimerBank (pga.mgh.harvard.edu/primerbank) or published papers. In this study, the relative expression of these genes in mice were detected: the reference gene: 18 s; the inflammatory genes: Cxcl1, Cxcl2, IL-6, Mcp1; the intestinal barrier-related genes: Zo-1 and Occludin; the gene of α1,2-fucosyltransferase: Fut2; the genes of taurine transporters:Slc6a6 and Slc36a1; the genes of taurine synthesis and metabolism genes: Cdo1, Csad, Fmo1, Ado, and Baat. The relative expression of these genes in E. coli were detected: the E. coli 16S rRNA and the kpsM gene. All primers used in this paper were listed in Table S2.

Measurement of intestinal permeability

Intestinal permeability was tested with Ussing Chambers (Physiologic Instruments, CA, USA) as previous described [38]. Briefly, the fresh colon segments (about 1 cm) were dissected 24 h after APAP injection, placed on a slide with their surfaces facing outward, and then mounted between the hemi-chambers of an Ussing apparatus. The colonic tissues were immersed in bilateral Krebs-Ringer solution, with a continuous flow of 5 % CO₂/95 % O₂ gas. The transepithelial potential and electricity were detected with two paired electrodes that contain 4 % agar in 3 M potassium chloride. The transepithelial resistance was calculated using Ohm's law.

Fluorescein isothiocyanate-dextran 4 kDa (FD4) powder (Sigma-Aldrich, St. Louis, Missouri, USA) was dissolved in Krebs solution to a concentration of 1 mg/mL. 2 mL of FD4 solution was added to the mucosal side of each half-chamber, while 2 mL of Krebs solution was added to the serosal side of each half-chamber. After measuring the fluorescence intensity at 520 nm in the serosal side of each half-chamber, we calculated the FD4 concentration, which represented the FD4 permeability.

Cell co-culture

The human normal intestinal epithelial cell line NCM460 cells were cultured in Dulbecco’s modified Eagle’s medium containing 4.5 g/L D-glucose (Gibco, CA, USA) and supplemented with 10 % heat-inactivated fetal bovine serum (Gibco, CA, USA) and 1 % penicillin–streptomycin (ServiceBio, Wuhan, China). The cells were incubated at a constant temperature (37 ℃) with 5 % CO₂. After the culture period, the NCM460 cells were harvested and seeded in 6-well plates (5 × 10⁵ cells per well) in medium without antibiotics. Once adherent, they were co-cultured with E. coli cell suspension at 37 ℃ for 2 h, and then collected cells for Western blotting.

Immunoprecipitation

Immunoprecipitation was performed using Immunoprecipitation Kit with Protein A + G Magnetic Beads (P2179M, Beyotime). Each 1.5 mg Dynabeads protein G (ThermoFisher Scientific) was bound with 5 µg of antibody (10 min, RT, gentle shaking) and incubated with 200 µl of EVs lysates (2 h at RT, gentle shaking). Protein elution was conducted with 20 µl elution buffer and 10 µl Western Blot loading buffer. After each step, beads were concentrated on magnetic particle concentrator and carefully washed with recommended washing buffer.

Western blotting

Proteins were extracted with RIPA lysis buffer (ServiceBio, Wuhan, China) supplemented with protease and phosphatase inhibitors. The protein concentration of each sample was determined by BCA Assay Kit (ServiceBio, Wuhan, China) and was adjusted to consistent concentration using lysis buffer. The proteins for Western blotting were added to the loading buffer at a ratio of 4:1and heated at 95 °C for 5 min. Then the samples were separated by SDS-PAGE, transferred to PVDF membranes, blocked, incubated with primary antibodies at 4 °C overnight and then with secondary antibodies. The relative expression of protein was quantified with ImageJ software. The antibodies used in this study were shown in Table S3.

ELISA assays

ELISA assays were carried out in accordance with the manufacturer’s protocol of ELISA kit. The following kits were used: LPS ELSIA Set (eBioscience), Human IL-6 ELISA Set (eBioscience), Taurine Assay Kit (Cell Biolabs, Inc.), Tauroursodeoxycholic acid (TUDCA) ELISA Set (Qingdao Jisskang Biotechnology Co, Ltd.).

Liquid chromatography-MS/MS

50 mg of mice liver specimens was mixed with 400 μl extraction solution, then it was grounded for 6 min (−10 °C, 50 Hz). The mixture was centrifuged at 13,000g for 15 min, and the supernatant was transferred to the injection vial for LC-MS/MS analysis. A pooled quality control sample (QC) was prepared by mixing all samples in equal volumes. The data were collected using a Thermo UHPLC-Q Exactive HF-X mass Spectrometer according to the operating instructions. The raw data was analyzed by Progenesis QI 2.3 (Water Corporation, Milford, USA) software for peak detection. Internal standard peaks were deleted from data matrix. At least 80 % of the metabolic features detected in any set of samples were retained, the maximum mass error allowed was ±10 ppm, only the metabolites with MS/MS fragments score above 30 were considered as confidently identified. Partial Least Squares Discriminant analysis (PLS-DA) was conducted to discriminate significant differences between the groups. Metabolites with consistent trends in each comparison group were used for subsequent analyses

Targeted metabolomics

A 20 mg tissue sample (including cecum content, liver, or plasma) was obtained and mixed with 2 μL of internal standard mixed working solution (10 μg/mL) and 198 μL of 20 % methanol–acetonitrile for homogenization. The mixture was then vortexed at 2500 r/min for 10 min and placed at −20 °C for an additional 10 min. Afterward, it was centrifuged at 4 °C for 10 min at 12,000 rpm. The supernatant was collected and passed through a protein precipitation plate before being prepared for LC-MS/MS analysis. The mass spectrometry conditions were: electrospray ionization temperature of 550 °C, mass spectrometry voltage of −4500 V, and curtain gas of 35 psi. In the triple quadrupole, each ion pair was scanned and detected based on optimized declustering potential and collision energy. A mixed solution was used as a QC sample, and one QC analysis sample was inserted every 10 monitored samples during the instrument loading process. By overlaying and analyzing the Total Ion Chromatogram (TIC) of the same QC sample, the stability of the instrument during the detection period could be assessed.

Data processing and statistical analysis

For mouse or cell experiments, the results of each group were presented as mean ± SEM. Shapiro-Wilk test was performed to assess the normality of data (Table S5). For data meeting the normality distribution, the comparison between two groups was performed using the independent samples t-test. For data not meeting this criterion, Wilcoxon rank sum test was employed for comparison. For the analysis and comparison of three or more groups, two-way Analysis of Variance (ANOVA) or Kruskal-Wallis test was employed, and the Tukey’s post-hoc test was used for multiple comparisons between groups. Statistical analysis and graphing were conducted using GraphPad Prism 9.0 software and R language.

Results

Gut dysbiosis was present in DILI patients

To determine the alterations of gut bacteria in DILI patients, full-length 16S sequencing was performed on fecal samples from DILI patients (DILI group, n = 42) and healthy controls (HC group, n = 34). Their clinical data were shown in Table S1. There were no significant differences in age, gender, body mass index, and blood pressure between the two groups. A total of 1175 OTUs were annotated. The DILI group exhibited 144 unique OTUs, the HC group demonstrated 163 unique OTUs, and a total of 868 OTUs were shared by the two groups (Fig. 1A). In terms of α-diversity, the Simpson index was higher and the Shannon index was lower in the DILI group, indicating that the diversity of gut bacteria in the DILI group was lower than that in the HC group (Fig. 1B). For β-diversity, PCoA analysis showed a significant separation between two groups (Fig. 1C). At the genus level, Escherichia and Streptococcus were up-regulated in the DILI group, conversely, Ruminococcus, Faecalibacterium, and Blautia exhibited down-regulation compared to the HC group (Fig. 1D). At the species level, E. coli was the top-ranked microorganism in the DILI group (Fig. 1E). We also cultured and identified E. coli from the feces of healthy individuals and DILI patients. The median number of E. coli per gram of feces was significantly higher in the DILI group compared to the HC group, which was consistent with the sequencing results (Fig. S1A and B). RDA, a method to visualize the correlation between bacterial communities and concerned factors, showed that Escherichia were positively correlated with TB, ALP, GGT, and ALT, but negatively related with ALB (Fig. 1F). Spearman's correlation analysis of clinical indices with the top10 strains of relative abundance revealed that only E. coli was significantly and positively correlated with TB and GGT (Fig. 1G). The levels of plasma LPS and IL-6 were also detected using ELISA, and they were increased in the DILI group (Fig. S1C). These results indicated that DILI patients had impaired intestinal barrier function and inflammatory dysregulation, which may be associated with increased levels of E. coli.

Fig. 1.

Gut dysbiosis was present in DILI patients. A Venn diagram of DILI group and HC group at the OTU level of 16S rRNA sequencing. B The indices of Shannon and Simpson at the OTU level of DILI and HC group (using Wilcoxon rank-sum test). C PCoA diagram of gut bacteria at the OTU level in the DILI group and HC group (using unweighted_unifrac distance algorithms, ANOSIM analysis compared the difference). D and E The circle diagram of gut bacteria at the genus and the species level. The left half of the circle represented the mean proportion of each bacterium in the DILI group or the HC group, while the right half of the circle represented the bacterium category and its average distribution ratio in each group. The relative abundance of E. coli in the HC group and the DILI group was compared using Wilcoxon rank-sum test. F The plot of redundancy analysis. The green arrows were the common clinical indicators of liver injury, like alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), etc. The blue arrows were bacteria. The longer the length of the arrow, the greater the influence of the indicator on the composition of gut microbiota. The red and blue dots represented the genera of DILI patients and healthy individuals, respectively. The closer the distance between the two points, the more similar the gut microbiota of the two samples were. The indicator was positively correlated with alterations in the microbial community of the sample if the point was located in the same direction as the arrow. G Spearman’s correlation analysis of liver function indicators with the relative abundance of gut bacteria at the species level. H The indices of Shannon and Simpson at the OTU level of mild group and moderate-to-severe group (using Wilcoxon rank-sum test). I PCoA diagram of gut bacteria at the OTU level in the mild group and moderate-to-severe group (using unweighted_unifrac distance algorithms, ANOSIM analysis compared the difference). J Bar plots of bacterial genera (ranked by mean abundance) in the mild group and moderate-to-severe group. K The relative abundance of E. coli in the mild group and moderate-to-severe group (using Wilcoxon rank-sum test). L The detection rates of the kpsM gene in the DILI group and the HC group, and in the mild group and moderate-to-severe group. HC group, n = 34, DILI group, n = 42. Mild group, n = 25, Moderate-to-Severe group, n = 17. * p < 0.05, ** p < 0.01, *** p < 0.001.

To further elucidate whether E. coli was involved in the progression of DILI, we classified DILI patients into the Mild group (n = 25) and the Moderate-to-Severe group (n = 17) based on the severity of liver damages. There were no significant differences between the two groups in terms of gender, age, blood pressure, BMI, DILI subtypes, etc., but liver injury indicators, such as TB, ALT, AST, and ALP were significantly higher in the Moderate-to-Severe group. With respect to α-diversity, the Simpson index was higher and the Shannon index was lower in the Moderate-to-Severe group, indicating that the diversity of intestinal bacteria in the Moderate-to-Severe group was lower than that in the Mild group (Fig. 1H). The difference in β-diversity between the two groups was also significant (Fig. 1I). The relative abundance of Escherichia and E. coli was notably higher in the Moderate-to-Severe group (Fig. 1J and K).

Previous studies suggested that the adhesion of E. coli to host cells is a crucial process in its pathogenicity. The CPS promoted their invasion and protected them from elimination by host immune cells. The kpsM gene, a key molecule in the transportation of CPS in E. coli, was often regarded as a virulence factor [18]. Given that E. coli ranked first in relative abundance (Fig. 1K) and showed a significant positive correlation with liver function (Fig. 1G), we hypothesized there might be different abundance of kpsM gene in E. coli between the Mild group and the Moderate-to-Severe group. The quantitative real-time PCR was then employed to detect the kpsM gene. The detection rate was 14.7 % in the HC group and significantly higher (54.8 %) in the DILI group, with 40.0 % in the Mild group and 76.5 % in the Moderate-to-Severe group (Fig. 1L). Thus, E. coli expressing kpsM gene may participate in exacerbating DILI.

E. coli expressing kpsM gene exacerbated AILI in mice

To explore the alterations of E. coli in mice with acute DILI, we administered APAP (300 mg/kg body weight) intraperitoneally to mimic liver injury (AILI model). Fecal samples were collected from mice before APAP injection and 24 h post-treatment for culture. The results revealed a significant increase in the level of fecal cultivable E. coli after APAP injection (Fig. S1D). Wild-type mice were gavaged with a cocktail of antibiotics (Abx) for five days to clear their gut microbiota, followed by intraperitoneal injection of APAP (Fig. S1E). The serum levels of ALT and AST were significantly lower in the Abx + APAP group compared to the PBS + APAP group (Fig. S1F). The above results indicated that AILI mice also exhibited gut dysbiosis, with an increase in the relative abundance of E. coli.

To validate the effect of feces from human patients and healthy donors, mice were gavaged with antibiotics to eliminate their gut bacteria. Subsequently, they received three consecutive days of fecal microbiota transplantation (FMT) using either healthy human or patient feces, and then they were injected with APAP (Fig. S2A). Mice received stools from healthy human effectively mitigated AILI in mice, while those received microbiota from DILI patients exacerbated liver injury and gut barrier dysfunction (Fig. S2B–D). These results indicated that fecal microbiota rich in E. coli contributed to the development of DILI.

To further study whether the kpsM gene of E. coli affected the progression of DILI, we constructed kpsM⁺ E. coli (EC^WT) and kpsM^- E. coli (EC^ΔkpsM), the sequence of kpsM gene of E. coli was showed in Table S4. The qPCR experiments confirmed successful knockout of the kpsM gene (Fig. S3A). Functional assays suggested that kpsM gene knockout reduced E. coli's phagocytosis resistance and adhesion capacity (Fig. S3B and C). Two hours after APAP, plasma lipopolysaccharide levels increased significantly, indicating the presence of intestinal barrier dysfunction (Fig. S3D). Thus, to determine whether E. coli expressing the kpsM gene could exacerbate DILI, mice were transplanted with EC^WT or EC^ΔkpsM two hours after APAP injection (Fig. 2A). The median number of cultivable E. coli per gram of mouse feces is comparable to that found in the feces of patients with DILI (Fig. S1B). In the APAP + EC^WT group, the serum levels of ALT and AST were significantly higher than the APAP + PBS group (Fig. 2B and C). The mRNA levels of the hepatic inflammatory cytokines, like Cxcl1 and Cxcl2, were also upregulated (Fig. 2D and E). HE staining of liver sections showed a significant increase in the necrotic area of liver lobes in the APAP + EC^WT group (Fig. 2F). However, in the APAP + EC^ΔkpsM group, the above indices were significantly lower than those in the APAP + EC^WT group (Fig. 2B–F). In the non-model groups, there were no significant differences in hepatic enzymes, inflammatory factors, or necrotic area between the CON + EC^WT group, the CON + EC^ΔkpsM group, and the CON + PBS group (Fig. 2B–F). We also explored the effect of intragastric administration EC^WT before the APAP injection (Fig. S4A). Compared to the PBS + APAP group, the EC^WT + APAP group resulted in a significant increase in ALT levels, while AST levels showed a trend toward worsening (Fig. S4B). In summary, E. coli alone was not sufficient to induce significant liver injury under normal physiological conditions, but it could exacerbate AILI especially following APAP-mediated intestinal barrier dysfunction, with the kpsM gene being a key pathogenic factor.

Fig. 2.

E. coli expressing kpsM gene exacerbated AILI in mice. A C57BL/6J mice were injected with acetaminophen, and two hours later, they were gavaged with PBS, EC^WT, or EC^ΔkpsM, respectively. B and C Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the CON + PBS, CON + EC^WT, CON + EC^ΔkpsM, APAP + PBS, APAP + EC^WT, and APAP + EC^ΔkpsM group, respectively. D and E Relative mRNA expression of hepatic Cxcl1 and Cxcl2 in liver tissues. F Representative pictures of H&E staining and quantification of necrotic areas in the liver (six plots shared a common scale bar = 100 μm). Mice were randomly divided into CON groups or APAP groups. Control groups: PBS, n = 7, EC^WT, n = 7, EC^ΔkpsM, n = 7. APAP groups: PBS, n = 12, EC^WT, n = 12, EC^ΔkpsM, n = 12. P values were determined by Two-way ANOVA with Tukeýs post-hoc test if not otherwise specified. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

E. coli expressing kpsM gene aggravated intestinal damage and promoted the intestinal FUT2 expression

To investigate how E. coli expressing the kpsM gene exacerbated AILI, we first focused on the intestinal inflammation and barrier function in mice. PCR results showed that the ileal inflammation indicators, such as IL-6 and Mcp1, were significantly higher in the APAP + PBS group compared to the CON + PBS group (Fig. 3A and B). The mRNA and protein levels of Occludin and Zo-1, indicators of intestinal barrier function, were also significantly decreased in the APAP + PBS group (Fig. 3C and D, Fig. S4C). These results indicated that APAP caused intestinal inflammatory damage and impaired barrier function, which was consistent with previous reports [39]. Compared to the APAP + PBS group, the APAP + EC^WT group further up-regulated the ileal inflammation and destroyed the intestinal barrier function, suggesting that E. coli carrying the kpsM gene could further exacerbate APAP-induced intestinal damage. In contrast, compared to the APAP + EC^WT group, the above indicators in the APAP + EC^ΔkpsM group were significantly reduced, almost indistinguishable from the APAP + PBS group, indicating that the loss of the kpsM gene attenuated the pathogenicity of E. coli (Fig. 3A–E). Additionally, there were no significant differences in the ileal inflammation and barrier function between the CON + EC^WT, CON + EC^ΔkpsM, and CON + PBS groups (Fig. 3A–E), indicating that E. coli had a low impact under physiological conditions with normal intestinal microbiota and intact barrier function. The permeability of colonic mucosa isolated from APAP-treated mice was further detected by Ussing chamber. The APAP + EC^WT group had lower transepithelial resistance and higher FD4 permeability compared to the APAP + PBS group, which was consistent with the results in the ileum (Fig. 3F). The plasma level of LPS in the APAP + EC^WT group was also higher than the APAP + PBS group and the APAP + EC^ΔkpsM group (Fig. 3G). Taken all together, E. coli carrying the kpsM gene could exacerbate APAP-induced intestinal injuries.

Fig. 3.

E. coli expressing kpsM gene aggravated intestinal damage and promoted the intestinal FUT2 expressions. A-D Relative mRNA expression of ileal IL-6, Mcp-1, Zo-1, and Occludin in the CON + PBS, CON + EC^WT, CON + EC^ΔkpsM, APAP + PBS, APAP + EC^WT, and APAP + EC^ΔkpsM group, respectively. E The ileal protein level of Zo-1 and Occludin in the AILI mice receiving different treatments. F The colonic barrier function in the AILI mice receiving different treatments. The larger the transepithelial electrical resistance (TEER) was, the more complete the barrier function was; however, the larger the fluorescein isothiocyanate-dextran 4 kDa (FD4) was, the higher the intestinal permeability was. G The level of serum lipopolysaccharide (LPS) in the different groups. H Relative mRNA expression of ileal Fut2 in the different groups. I The protein level of intestinal Fut2 in the AILI mice receiving different treatments. J The UEA-I staining of terminal ileum in the AILI mice receiving different treatments, scale bars: first row = 200 μm, second row = 50 μm. K The protein level of Fut2 in the NCM460 cells co-culturing with EC^WT or EC^ΔkpsM. Mice were randomly divided into CON groups or APAP groups. Control groups: PBS, n = 7, EC^WT, n = 7, EC^ΔkpsM, n = 7. APAP groups: PBS, n = 12, EC^WT, n = 12, EC^ΔkpsM, n = 12. P values were determined by Two-way ANOVA with Tukeýs post-hoc test if not otherwise specified. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Previous studies have reported that the intestinal FUT2 contributes to the attachment and survival of E. coli, and inhibiting the gene expression can enhance the host's resistance to E. coli [31]. We therefore explored the impact of E. coli on Fut2 gene expression. In the CON groups, neither type of E. coli could up-regulate Fut2 (Fig. 3H). In the APAP model groups, E. coli expressing the kpsM gene was able to increase the mRNA and protein level of Fut2, while E. coli without the kpsM gene was not (Fig. 3H and I). UEA-I staining revealed that α1,2-fucosylation was increased in the ileum of APAP + EC^WT mice. (Fig. 3J). We also co-cultured the NCM460 cell with E. coli and detected the expression of Fut2, and the result was consistent (Fig. 3K). Additionally, there was no significant difference in Fut2 expression between the CON + PBS group and the APAP + PBS group. However, its expression in the APAP + EC^WT group was significantly up-regulated compared to the CON + EC^WT group. This might be that APAP itself had limited influence on the host Fut2 expression, and that the host was more sensitive to the stimulation of EC^WT after APAP-induced intestinal damage. To sum up, the intestinal Fut2 gene might be an important host target in the exacerbation of AILI by E. coli expressing the kpsM gene.

Intestinal epithelial Fut2 knockout ameliorated the aggravation of E. coli on AILI

To validate the conclusion above, we used intestinal epithelial cell-specific Fut2 gene knockout mice, which was verified in our previous paper [30]. They were administered APAP intraperitoneally to mimic acute severe DILI, and then gavaged by E. coli or PBS (Fig. 4A). The liver injury (ALT and AST), the mRNA levels of hepatic inflammatory cytokines (Cxcl1 and Cxcl2), and the area of hepatic necrosis in the EC^WT + Fut2^ΔIEC group were significantly decreased compared to the EC^WT + Fut2^fl/fl group. The EC^ΔkpsM gavage did not exacerbate liver injury in the Fut2^ΔIEC or Fut2^fl/fl groups, which was consistent with the results of wild-type mice (Fig. 4B–F). These findings confirmed that knocking out the intestinal Fut2 gene could significantly ameliorate the exacerbation of AILI by E. coli expressing the kpsM gene, but the harmful effects of Fut2 seemed to be limited in the model built using APAP alone.

Fig. 4.

Intestinal epithelial Fut2 knockout ameliorated the aggravation of E. coli on AILI. A Fut2^fl/fl or Fut2^ΔIEC mice were injected with acetaminophen, and two hours later, they were gavaged with PBS, EC^WT, or EC^ΔkpsM, respectively. B and C Serum ALT and AST in the Fut2^fl/fl groups and the Fut2^ΔIEC groups treated with PBS, EC^WT, or EC^ΔkpsM, respectively. D and E Relative mRNA expression of hepatic Cxcl1 and Cxcl2 in liver tissues. F Representative pictures of H&E staining and quantification of necrotic areas in the liver (scale bar = 100 μm). APAP + Fut2^fl/fl groups: PBS, n = 5, EC^WT, n = 8, EC^ΔkpsM, n = 5. APAP + Fut2^ΔIEC groups: PBS, n = 5, EC^WT, n = 9, EC^ΔkpsM, n = 5. P values were determined by Two-way ANOVA with Tukeýs post-hoc test if not otherwise specified. * p < 0.05, ** p < 0.01, *** p < 0.001.

Knockout of intestinal epithelial Fut2 affected the hepatic metabolite profiles

Previous studies have shown that the level of Fut2 affects the carbohydrate and lipid metabolism [40]. To clarify the mechanisms of Fut2 exacerbating the development of AILI, we analyzed the hepatic metabolite profiles of Fut2^fl/fl and Fut2^ΔIEC mice after administration of APAP + EC^WT using untargeted metabolomics.

OPLS-DA is a supervised multivariate statistical method commonly used to distinguish differences in metabolic profiles among groups. For hepatic metabolites, there was also a significant separation between the two groups (Fig. 5A). 90 metabolites were up-regulated and 48 metabolites were down-regulated in the Fut2^ΔIEC group (Fig. 5B). The KEGG enrichment analysis revealed that the primary bile acid biosynthesis, the pentose phosphate pathway, etc., were main distinct pathways (Fig. 5C). The network interaction and relative levels of those metabolites were showed in Fig. 5D and E.

Fig. 5.

Knockout of intestinal epithelial Fut2 affected the hepatic metabolite profiles. A–C The OPLS-DA plot, volcano plot, and KEGG enrichment analysis of hepatic metabolites in the Fut2^fl/fl and the Fut2^ΔIEC mice treated with APAP and EC^WT. D The network diagram of KEGG pathways and related metabolites. E The relative abundance of taurine and other enriched metabolites from the Fig. 5D in the Fut2^fl/fl and the Fut2^ΔIEC mice treated with APAP and EC^WT. F The level of taurine-related bile acids in the liver. For data meeting the normality distribution, independent samples t-tests were used for comparisons between two groups; for data not meeting this criterion, Wilcoxon rank-sum test was conducted. Fut2^fl/fl group, n = 8; Fut2^ΔIEC group, n = 9. * p < 0.05. ** p < 0.01.

Knockout of intestinal epithelial Fut2 up-regulated the tauroursodeoxycholic acid

Previous studies have shown that the deletion of the intestinal Fut2 gene affects the bile acid metabolism [27]. In this study, hepatic differential metabolites were also enriched in the primary bile acid synthesis pathway. Specifically, the relative abundance of taurine in the Fut2^ΔIEC group was more than twice that of the Fut2^fl/fl group (Fig. 5E), representing the largest increase among the enriched metabolites shown in Fig. 5D. Previous studies revealed that taurine could alleviate AILI in rats [41]. We designed four groups of animal experiments to validate the efficacy of taurine: PBS + APAP, taurine + APAP, PBS + APAP + EC^WT, taurine + APAP + EC^WT. The mice were administered taurine (300 mg/kg body weight) or PBS via oral gavage before the APAP injection (Fig. S5A). The liver injury indicators, ALT and AST, were decreased significantly in taurine + APAP + EC^WT group compared with the PBS + APAP + EC^WT group, indicating that supplementation of taurine could alleviate AILI (Fig. S5B).

Given that taurine can further synthesize secondary bile acids [41,42], we conducted targeted metabolomics of 13 taurine-related bile acids in the liver. Tauroursodeoxycholic acid (TUDCA) was the only molecule significantly up-regulated in the Fut2^ΔIEC group, with an average level 6.98 times that of the Fut2^fl/fl group (Fig. 5F). TUDCA is produced mainly through the conjugation of taurine and ursodeoxycholic acid in vivo and has choleretic and anti-inflammatory properties, which can improve AILI [43,44]. Besides, we also detected the levels of taurine and TUDCA in the mouse livers using ELISA. Whether in the physiological state, AILI condition, or severe AILI exacerbated by E. coli, both of taurine and TUDCA in the Fut2^ΔIEC group were higher than those in the Fut2^fl/fl group (Fig. S6A and B). These results suggested that intestinal epithelial Fut2 knockout improved the exacerbating effect of E.coli on AILI, which might be highly correlative with up-regulation of hepatic taurine and TUDCA levels.

Fut2 did not affect the mRNA expression of genes related to the taurine transport and synthesis

Next, we tried to find the reason for the increase of taurine and TUDCA in the liver. In humans and mice, taurine is mainly derived from intestinal absorption and hepatic synthesis (Fig. 6A). The main proteins in charge of taurine transport in the intestine are taurine transporter (TauT, encoded by the Slc6a6 gene) and proton-coupled amino acid transporter (PAT1, encoded by the Slc36a1 gene). In the liver, there are three pathways for synthesizing endogenous taurine. The cysteinesulfinate route is the main pathway, with cysteine dioxygenase (CDO, encoded by the Cdo1 gene) and cysteine sulfinic acid decarboxylase (CSAD, encoded by the Csad gene) as the key catalytic enzymes. The alternative pathway is the conversion of L-cysteine to cysteamine, which can be catalyzed by cysteamine dioxygenase (ADO, encoded by the Ado gene). Ultimately, flavin-containing monooxygenase 1 (FMO, encoded by the Fmo1 gene) catalyzed the conversion of hypotaurine to taurine. Taurine is then used for conjugation of bile acids, such as taurocholate catalyzed by bile acid-CoA: amino acid N-acyltransferase (BAAT, encoded by the Baat gene) [45]. Hence, there were other two possible causes for its intrahepatic increase: one was the increase in intestinal transport and the other was the increase in hepatic synthesis. We subsequently tested the transcription of above molecules.

Fig. 6.

Fut2 inhibited the absorption of taurine through fucosylating TauT. A The absorption and metabolism process of taurine in mammals. B and C Relative mRNA expression of Slc6a6 and Slc36a1 in different intestinal parts. D Relative mRNA expression of hepatic enzymes in the taurine synthesis and metabolism. E Fut2^fl/fl or Fut2^ΔIEC mice were gavaged with a high dose of taurine (300 mg/kg), and specimens were collected three hours later. F The content of taurine in the cecum content, liver, or plasma in the Fut2^fl/fl group (n = 8) and the Fut2^ΔIEC group (n = 8). G The potential glycosylated site of TauT in mouse and human. H Immunoblotting for the indicated molecules in the Caco2 cells lysate that were precipitated with the anti-Fut2 antibody. For data meeting the normality distribution, independent samples t-tests were used for comparisons between two groups; for data not meeting this criterion, Wilcoxon rank-sum test was conducted. Fut2^fl/fl group, n = 8; Fut2^ΔIEC group, n = 8–9. * p < 0.05, **** p < 0.0001.

There were no obvious differences in mRNA levels Slc6a6 and Slc36a1 between the Fut2^fl/fl group and the Fut2^ΔIEC group treated with APAP and EC^WT, either in the jejunum, ileum, or colon (Fig. 6B and C). Hepatic taurine synthase including Cdo1, Csad, Ado, and Fmo1, and bile acid synthase Baat were not significantly altered, either (Fig. 6D). These results suggested that knockout of intestinal epithelial Fut2 was unlikely to affect the expression of taurine transport genes in the gut and taurine synthetic genes in the liver.

Fut2 inhibited the absorption of taurine through fucosylating TauT

We then explored whether the transport of taurine was affected by intestinal Fut2. The Fut2^ΔIEC and Fut2^fl/fl mice fed in the same cage were gavaged after 24 h fasting, the taurine level was then measured in the cecum, liver, and blood after 3 h gavage based on the whole gut transit time (Fig. 6E). The targeted-metabolomic results showed that the plasma and hepatic level of taurine in Fut2^ΔIEC mice was higher than Fut2^fl/fl mice, but the cecal taurine was lower than Fut2^fl/fl mice (Fig. 6F). These results suggested that intestinal epithelial Fut2 knockout might promote the transport of taurine from the gut to the liver.

Considering that Fut2 is responsible for alpha1,2-fucosylation, which attaches fucose to the asparagine of a protein in an N-linked manner, we hypothesized that Fut2 could modify TauT. The UniProt database showed that the asparagine site was present in both human (P31641) and murine (O35316) TauT protein sequences. The predicted N-glycosylation sites were at amino acid 163, 179, and 190 (Fig. 6G). Moreover, immunoprecipitation assays suggested that Fut2 was associated with TauT in Caco2 cells (Fig. 6H). These results suggested that the protein may undergo fucosylation modification which in turn affected its function.

The plasma level of taurine decreased in Moderate-to-Severe DILI patients

To further validate the findings observed in mice, this study collected plasma samples from DILI patients for untargeted metabolomics analysis. Patients with plasma specimens were categorized into the Mild group (n = 24) and the Moderate-to-Severe group (n = 12). The OPLS-DA showed a significant separation between the two groups, with the first principal component explaining 10.80 % of the variance (Fig. 7A). 94 metabolites were up-regulated and 158 were down-regulated in the Moderate-to-Severe group (Fig. 7B). KEGG enrichment analysis revealed that they were mainly enriched in the bile secretion pathway, the primary bile acid biosynthesis pathway, and the cholesterol metabolism pathway (Fig. 7C). A total of 12 molecules belonged to the primary bile acid biosynthesis pathway. Compared to the Mild group, the levels of glycocholic acid (GCA) and 5b-cyprinol sulfate were increased in the Moderate-to-Severe group, while taurine, tetrahydrocortisol, cholestane-3, 7, 26-triol, 3α,7α,26-trihydroxy-5β-cholestane, and 3α,7α-dihydroxy-5β-cholestan-26-ol were significantly decreased (Fig. 7D). Spearman correlation analysis revealed that taurine was negatively correlated with ALP and TB, but positively correlated with ALB (Fig. 7E). Given that bile acid metabolism is dependent on the gut microbiota, we also analyzed the relationship between E. coli and taurine. The E. coli count per gram of human feces was negatively associated with the level of taurine (Fig. 7F). To sum up, the disruption of taurine metabolism also exists in DILI patients, and the more severe the DILI, the lower the level of taurine is.

Fig. 7.

The plasma level of taurine decreased in Moderate-to-Severe DILI patients. A–C The OPLS-DA plot, volcano plot, and KEGG enrichment analysis of plasma metabolites in mild and moderate-to-severe DILI patients. Mild group, n = 24, Moderate-to-Severe group, n = 12. D The relative abundance of metabolites in the primary bile acids biosynthesis pathway. E The Spearman correlation heatmap of clinical indicators and plasma metabolites. ALP, alkaline phosphatase, TB, total bilirubin, DB, direct bilirubin, ALB, albumin, AST, aspartate aminotransferase, GLB, globulin, ALT, alanine aminotransferase, GGT, γ-glutamyl transferase. F The relationship between the plasma level of taurine and the number of cultivable E. coli in DILI patients, n = 29. For data meeting the normality distribution, independent samples t-tests were used for comparisons between two groups; for data not meeting this criterion, Wilcoxon rank-sum test was conducted. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Discussion

E. coli is a Gram-negative bacterium that is widely found in the intestinal tracts of humans and animals. Most of them are commensal, but some strains develop into opportunistic pathogens through the acquisition of virulence genes or antibiotic resistance genes, which can cause intestinal and extra-intestinal infections, such as urinary tract infections, meningitis, and sepsis [46]. Several recent studies have demonstrated that E. coli can exacerbate hepatic injury by disrupting the intestinal epithelial barrier and vascular barrier [16], activating the Toll-like receptor 4 and promoting the release of pro-inflammatory factors [47,48], inducing mesenchymal transformation of liver sinusoidal endothelial cell [15] and disrupting bile acid metabolism [49]. Our study revealed a gradual increase in fecal E. coli abundance as DILI progressed, tentatively suggesting that E. coli might be associated with the development of DILI.

CPS is an important virulence factor of E. coli, which is usually located in the outer layer of the bacterial cell wall [50]. On the one hand, CPS helps E. coli adhere to the host intestinal mucosa and promotes its further colonization and infection [51]; on the other hand, CPS protects the bacteria from the host immune system attack, especially from macrophage phagocytosis, which makes E. coli difficult to be removed and increases its ability to survive and spread in the host body [52]. The kpsM gene belongs to the ATP-Binding Cassette (ABC) transporter protein superfamily in E. coli [53], which constitutes a Type II CPS export machinery together with kpsE, kpsT, and other genes [17]. The primary function of this system is to transport newly synthesized CPS from the cytoplasm to the cell surface by hydrolyzing adenosine triphosphoric acid for energy. Deletion of the kpsM gene leads to increased susceptibility of E. coli to host immune responses, thereby reducing its survival and pathogenicity [36,53]. Under normal intestinal microbiota and intact barrier function, kpsM⁺ E. coli are easily eliminated by the body's immune system. However, during DILI states, immune dysfunction and disruption of the gut barrier reduce resistance to kpsM⁺ E. coli, making them more likely to aggravate DILI progression. A prior study revealed that kpsM⁺ E. coli was enriched in patients with alcohol-associated liver disease (ALD), and the presence increased patient mortality. This strain primarily evades recognition and clearance by hepatic macrophages through the formation of a capsule, thereby facilitating its own proliferation and dissemination. Inhibiting kpsM gene of E. coli can improve ALD in mice [54]. This study further clarified that the detection rate of the kpsM gene was higher in patients with moderate-to-severe DILI, and verified in vivo that E. coli expressing kpsM gene could worsen AILI through affecting the host's intestinal epithelial Fut2 and bile acid metabolism, while E. coli lacking this gene did not exhibit this effect. These findings deepened the understanding of the mechanisms of gut microbiota’s influence on DILI to the genetic level of strains.

Fucosylation, a prevalent post-translational modification, involves the attachment of a fucose molecule to a glycoprotein. For example, fucosyltransferase 8 catalyzes core fucosylation [55]. Fut2 primarily catalyzes alpha1,2-fucosylation, which attaches L-fucose to the terminal galactose of the sugar chain. It is generally accepted that the inhibition of Fut2 exacerbates intestinal inflammation [29,30,56,57]. However, the results of Fut2 studies on hepatic injury are contradictory. Fut2 deficiency or inhibition of α1,2-fucosylation exacerbates alcohol-associated liver injury [58], yet ameliorates steatohepatitis [27] and AILI [59]. Based on the previous reports, this study employed intestinal epithelium-specific knockout Fut2 mice and confirmed that the inhibition of α1,2-fucosylation mitigated the exacerbating effect of E. coli on AILI, which provided new evidence for the modulation of intestinal fucosyltransferase activity for the treatment of DILI.

The interaction between E. coli and fucosyltransferase has also been reported. On the one hand, the level of Fut2 affects the composition and structure of gut microbiota, especially E. coli. A clinical study found that loss-of-function mutations in the Fut2 gene resulted in a reduction of Escherichia in fecal samples [28]. In turn, E. coli also influences the host fucosyltransferase expression. For instance, enterotoxigenic E. coli F18 can up-regulate the Fut8 expression in pigs [60]. In addition, low levels of Fut2 or Fut8 conferred host resistance to E. coli [60,61]. In this study, we discovered that E. coli expressing the kpsM gene, but not those with the gene knocked out, were able to up-regulate the host intestinal expression of Fut2, which added new evidence for E. coli and host interactions.

Taurine is a sulfur-containing semi-essential amino acid that is widely distributed in mammalian tissues, with higher levels found in the liver, brain, and heart. Existing research suggests that the substance exerts a protective role within the body. In the gut, taurine can enhance barrier function [62,63], attenuate intestinal inflammatory responses [64], reduce intestinal oxidative stress [65], and protect intestinal epithelial cells from oxidative damage [66]. In the liver, taurine alleviates cholestatic liver disease by binding to bile acids, which are less toxic and more easily excreted [67]. Taurine can also indirectly reduce oxidative stress and ameliorate AILI by enhancing glutathione synthesis [41]. Moreover, bile acids that bind taurine possess a multitude of protective effects, such as anti-inflammation, antioxidation, and inhibition of endoplasmic reticulum stress. For example, TUDCA has been approved by FDA for the treatment of cholestatic liver disease [68]. In our study, the hepatic levels of taurine and TUDCA of Fut2 knockout mice were significantly higher than those of the control group. The clinical study also observed that the plasma taurine level in patients with moderate-to-heavy DILI were indeed lower than those in patients with mild DILI, which indicated that taurine played an important role in the progression of DILI.

In conclusion, we clarified that kpsM⁺ E. coli was able to up-regulate the expression of the Fut2 gene in the intestinal epithelium cells, affecting the absorption of taurine, which led to a decrease in the hepatic taurine and affected the synthesis of TUDCA, eventually exacerbating AILI. In addition, plasma untargeted metabolomics of DILI patients verified the above findings again. The plasma taurine levels in patients with moderate-to-severe DILI were significantly lower than those in patients with mild DILI. In the future, detecting the relative abundance of E. coli in human feces, particularly identifying kpsM⁺ strains, may be helpful for early prediction of prognosis for DILI patients. Therapeutically, developing novel interventions, like kpsM⁺ E. coli-targeted phages, inhibitors of Fut2 protein or kpsM gene, may potentially contribute to the treatment of DILI, though specific effectiveness requires validation through clinical trials.

There were still some limitations in this study. First, given that the physical condition of patients with severe DILI was difficult to tolerate colonoscopic biopsy, it was unknown whether the colonic level of FUT2 protein was higher than that of patient with mild DILI. Second, given the complexity and diversity of the gut microbiota, whether and how other abundant bacteria in the intestines of DILI patients (such as Faecalibacterium prausnitzii) influence the onset and progression of DILI warrants further investigation. Third, how the kpsM⁺ E.coli up-regulates the intestinal Fut2 expression was also not clear. The underlying mechanisms of E. coli and their potential for clinical application also requires validation through large-scale clinical studies. Besides, the effects of Fut2 on the TAUT protein, such as its glycosylation sites, require further validation.

Data availability statement

Further information and requests for resources and data should be directed to the lead investigator, Huikuan Chu (2012XH0827@hust.edu.cn). This study does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead investigator upon reasonable request.

Funding sources

This study was supported by National Key R&D Program of China (No. 2022YFA1305600 to H.C, 2023YFC2413804 to L.Y), the National Nature Science Foundation of China (82470584, 82000561 to H.C, 82270614, 81974078, and 81570530 to L.Y), and the Science Foundation of Union Hospital (2021xhyn005 to H.C).

Compliance with ethics requirements

The clinical research protocol was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, and registered on ClinicalTrials (https://clinicaltrials.gov, NCT05465642). All subjects participating in this project signed informed consent forms.

Author contribution

H.C. and L.Y. designed this research and critically revised the paper. W.G. drafted the manuscript, analyzed and visualized the data. W.G., S.Y., and L.Z. performed the animal experiments. L.C., J.C., W.H., Y.C., A.L., Y.Z., Y.Y., participated in the patient recruitment and specimen collection. Z.P. and C.T. were responsible for purification and cultivation of bacteria. B.S. and X.H. edited and polished the manuscript. All authors reviewed and approved the manuscript. The authors have no conflict of interest related to this publication.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: