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. 2023 Dec 4:14:1276951.
doi: 10.3389/fmicb.2023.1276951. eCollection 2023.

Insight into the mechanism of gallstone disease by proteomic and metaproteomic characterization of human bile

Xue-Ting Yang #  1 , Jie Wang #  1 , Ying-Hua Jiang #  1 , Lei Zhang  1 , Ling Du  2 , Jun Li  3 , Feng Liu  1
Affiliations

Insight into the mechanism of gallstone disease by proteomic and metaproteomic characterization of human bile

Xue-Ting Yang et al. Front Microbiol. .

Abstract

Introduction: Cholesterol gallstone disease is a prevalent condition that has a significant economic impact. However, the role of the bile microbiome in its development and the host's responses to it remain poorly understood.

Methods: In this study, we conducted a comprehensive analysis of microbial and human bile proteins in 40 individuals with either gallstone disease or gallbladder polyps. We employed a combined proteomic and metaproteomic approach, as well as meta-taxonomic analysis, functional pathway enrichment, and Western blot analyses.

Results: Our metaproteomic analysis, utilizing the lowest common ancestor algorithm, identified 158 microbial taxa in the bile samples. We discovered microbial taxa that may contribute to gallstone formation, including β-glucuronidase-producing bacteria such as Streptococcus, Staphylococcus, and Clostridium, as well as those involved in biofilm formation like Helicobacter, Cyanobacteria, Pseudomonas, Escherichia coli, and Clostridium. Furthermore, we identified 2,749 human proteins and 87 microbial proteins with a protein false discovery rate (FDR) of 1% and at least 2 distinct peptides. Among these proteins, we found microbial proteins crucial to biofilm formation, such as QDR3, ompA, ndk, pstS, nanA, pfIB, and dnaK. Notably, QDR3 showed a gradual upregulation from chronic to acute cholesterol gallstone disease when compared to polyp samples. Additionally, we discovered other microbial proteins that enhance bacterial virulence and gallstone formation by counteracting host oxidative stress, including sodB, katG, rbr, htrA, and ahpC. We also identified microbial proteins like lepA, rtxA, pckA, tuf, and tpiA that are linked to bacterial virulence and potential gallstone formation, with lepA being upregulated in gallstone bile compared to polyp bile. Furthermore, our analysis of the host proteome in gallstone bile revealed enhanced inflammatory molecular profiles, including innate immune molecules against microbial infections. Gallstone bile exhibited overrepresented pathways related to blood coagulation, folate metabolism, and the IL-17 pathway. However, we observed suppressed metabolic activities, particularly catabolic metabolism and transport activities, in gallstone bile compared to polyp bile. Notably, acute cholelithiasis bile demonstrated significantly impaired metabolic activities compared to chronic cholelithiasis bile.

Conclusion: Our study provides a comprehensive metaproteomic analysis of bile samples related to gallstone disease, offering new insights into the microbiome-host interaction and gallstone formation mechanism.

Keywords: bile; cholecystitis; gallstone; metaproteomics; microbiome; proteomics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Proteomic and metaproteomic analysis of human bile with or without gallstone. (A) The cohort enrolled in this study contains 14 acute CHLs, 17 chronic CHLs and 9 gallstone-free patients with gallbladder polyps. The bile fluids were collected during gallbladder resection of gallstone patients or polyp remove of patients. The proteins were extracted and separated using SDS-PAGE, followed by in-gel tryptic digestion. LC–MS analysis was performed using TIMS-TOF pro mass spectrometer. (B) The peptides were identified by two-step database searching using Metalab against a comprehensive protein database (UniProtDB) and the peptides were further subjected to taxonomic lowest common ancestor (LCA) analysis. Metataxonomic analysis using public 16S rRNA datasets of human bile by Kraken2 program was performed independently to verify the metaproteomic results. (C) The correlation plot of the bile samples based on protein identification. (D) PLS-DA of bile samples based on identified microbial taxa. The ellipses illustrated the confidence interval.
Figure 2
Figure 2
Identification of microbes from gallstone and polyp bile samples. (A) The proportion of unique peptides identified in human, fungi, bacteria, archaea, and virus. (B) The structure and abundance of the top 10 phyla in both polyp and gallstone bile samples were analyzed using the intensity of identified microbial peptides. CHL, cholecystitis. (C) The structure and abundance of the top 10 microbial species in polyp and gallstone bile samples. (D) The taxa we identified were found in other Human body sites through data mining of public references. The details were deposited in Supplementary Table S2. (E) The tandem MS spectrum of peptide LYCEEVGWVICGIK identified from lepA protein (LEPA_BUCCC) of Buchnera aphidicola. (F) The Sankey plot shows the metataxonomic analysis of human bile samples. The dataset was retrieved from GenBank with the BioProject accession number PRJNA580086 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA580086). In this project, 45 biliary bile samples were analyzed using 16S rRNA sequencing. We re-analyzed the dataset against kraken2-microbial database (https://lomanlab.github.io/mockcommunity/mc_databases.html) using kraken2 program (Lu et al., 2022). The red arrowhead represents the clade of Buchnera aphidicola, which has been detected in 44 out of 45 bile samples, comprising a total of 7,313 reads.
Figure 3
Figure 3
Taxon changes associated with gallstone formation determined by the microbial peptides. (A) Hierarchical cluster analysis of the microbial taxa significantly changed in gallstone bile samples (n = 31) comparing to polyp bile samples (n = 9). (B,C) Linear discriminant analysis Effect Size (LEfSe) analysis of microbial biomarkers of CHL (n = 31) and polyp (n = 9) using an LDA threshold of 2. The numbers indicate the major marker clades in CHL. (D) The abundance of Helicobacteraceae between CHL (n = 31) and polyp (n = 9) patients based on the proteomic data. The p values were calculated using Welch’s t test. MS, mass spectrometry. (E) The abundance of Helicobacteraceae between CHL (n = 14) and healthy (n = 13) patients based on the 16S rRNA data (dataset PRJNA439241). 16S, 16S rRNA dataset analysis. Chole, cholecystitis. Healt, healthy.
Figure 4
Figure 4
The microbial proteins identified from gallstone and polyp bile samples. (A) Abundance rank of identified proteins. The intensity of each protein was determined by summing up the LFQ values across all samples. Microbial proteins were highlighted. (B) The abundance of differential microbial proteins between CHL (n = 31) and polyp (n = 9) patients. (C) The differential microbial proteins between acute CHL (n = 14) and polyp (n = 9) patients. (D) The differential microbial proteins between chronic CHL (n = 17) and polyp (n = 9) patients. (E) Three microbial proteins upregulated in CHL compared to polyp. p values were calculated using Welch’s t test. (F) The differential microbial proteins between acute CHL (n = 14) and chronic CHL (n = 17) patients.
Figure 5
Figure 5
The host proteins identified from gallstone and polyp bile samples. (A) Gene ontology (GO) enrichment was performed on the upregulated human proteins in CHL compared to polyp using the g:Profiler tool (https://biit.cs.ut.ee/gprofiler/gost) (Raudvere et al., 2019). (B) Differential expression of complement proteins between CHL and polyp samples. p values were calculated using unpaired Welch’s t test. (C) Two mucin proteins were upregulated in CHL compared to polyp. (D) GO enrichment of the downregulated human proteins in CHL compared to polyp. GO_MF, Gene ontology of molecular function; GO_CC, cellular component; GO_BP biological process; REAC, Reactome; WP, WikiPathways. (E) Proteins associated with bile secretion were downregulated in CHL compared to polyp.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The work was supported by the National Natural Science Foundation of China (NSFC) (No. 81572833 and 32000505).