Genomic and functional characterization of a mucosal symbiont involved in early-stage colorectal cancer

Abstract

Colorectal cancer is a major health concern worldwide. Growing evidence for the role of the gut microbiota in the initiation of CRC has sparked interest in approaches that target these microorganisms. However, little is known about the composition and role of the microbiota associated with precancerous polyps. Here, we found distinct microbial signatures between patients with and without polyps and between polyp subtypes using sequencing and culturing techniques. We found a correlation between Bacteroides fragilis recovered and the level of inflammatory cytokines in the mucosa adjacent to the polyp. Additional analysis revealed that B. fragilis from patients with polyps are bft-negative, activate NF-κB through Toll-like receptor 4, induce a pro-inflammatory response, and are enriched in genes associated with LPS biosynthesis. This study provides fundamental insight into the microbial microenvironment of the pre-neoplastic polyp by highlighting strain-specific genomic and proteomic differences, as well as more broad compositional differences in the microbiome.

Keywords: Bacteroides fragilis; colon cancer; colorectal polyp; inflammation; innate immunity; microbiome; microbiota.

Figure 1.. Compositional dysbiosis and enrichment for Bacteroides fragilis in patients with colorectal polyp

(A–C) (A) Hyperproliferation and inflammation marker expression revealed by immunohistochemistry (IHC) in polyp biopsies and PF control mucosal biopsies, no quantitative data available due to tissue restriction (B and C). Levels of cytokines in mucosal biopsy tissue samples detected by ELISA. Data are represented as the mean ± SEM of 3–4 independent experiments. (A–D), *p < 0.05. ***p < 0.001. One-way ANOVA with multiple comparisons. Levels are measured in a subset of 4 TAP patients, 2 SSP, and 3 PF patients. (D) Phylum-level abundance in 16S rRNA sequencing of 6 PA and 6 NPA TAP biopsies, 3 PA and 3 NPA SSP biopsies, and 6 PF biopsies. (E) Principal Coordinate of Analysis (PCoA) of microbiota composition by 16S at phylum level. Statistics are analysis of molecular variation (AMOVA) and homogeneity of molecular variance (HOMOVA). (F and G) Linear discriminant analysis (LDA) of phylum-level abundance in 16S of mucosal biopsies in TAP NPA, SSP NPA, and PF biopsies. (H) Heatmap representation of genus level abundance after 16S rRNA profiling. (I) Phylum level abundance in culturomics on 14 PA and NPA TAP biopsies, 10 PA and NPA SSP biopsies, and 9 PF biopsies. (J) Heatmap representation of genus level abundance in culturomics. (K) Species level abundance of Bacteroides species in culturomics. See also Figures S2–S4.

Figure 2.. NTBF recovery correlates with inflammation in host tissue

(A and B) Correlation between mucosal IL-12p40 and IL-10 concentration (pg/ml) and abundance of B. fragilis isolated in vitro. Statistics are Pearson correlation coefficient of all data points with each data point representing a biological replicate of NTBF isolate recovered from seven PA and seven NPA TAP biopsies (o), 4 PA and 3 NPA SSP biopsies (Δ), and 7 PF biopsies (•). (C) Correlation between polyp size and abundance of NTBF isolated. Statistics are Pearson correlation coefficient of all data points, each data point representing a biological replicate of NTBF isolate recovered from TAP (o) or PF (•) biopsy tissue. (D) Whole genome assembly and reconstitution of 39 B. fragilis clinical isolates from, 9 PA and 76 NPA biopsies from 4 TAP patients (Patient 3, 9, 33, and 37), 6 PA and 3 NPA isolated from 2 SSP patients (Patient 2 and 28), 9 PF isolates from 3 different patients (Patient 16, 27, and 36), and 2 PA and 3 NPA isolates from an HP (Patient 13). Patients positive for PSA gene (o) and bft (peach open box). Each patient involved in the analysis is labeled with a specific color on the tree, the tree also features the 15 PA (dark green star) and 10 NPA (Light green star) isolates recovered from 4 TAP (blue dot), 2 SSP (peach dot), 1 HP (black dot), and 9 PF (grey dot). (E) Genome reconstitution of the B. fragilis clinical isolates featuring PA (green box) versus NPA (peach box) from TAP (blue box), SSP (pink box), HP (black box), and PF (grey box). (F) Venn diagram of single nucleotide variants (SNVs) in the genome of the PA and NPA from TAP, SSP, and PF patients. (G) Heatmap representing the proportion of virulence genes present in the genome of PA and NPA B. fragilis isolates from TAP, SSP, and PF patients.

Figure 3.. NTBF isolates from patients with polyps induce inflammation in vitro and signal through TLR4

(A–D) Levels of cytokines detected from co-culturing Bacteroides fragilis supernatants with monocytic cell line by ELISA. Data are represented as the mean ± SEM of 3–4 independent experiments including 16 PA and 16 NPA isolates from 4 different TAP patients, 8 PA and 8 NPA clinical isolates from 2 different SSP patients, and 4 clinical isolates from 1 PF patient. ETBF+ represents the bft positive study isolates that include 4 PA and 4 NPA isolates from 1 TAP patient and 8 isolates from 2 PF patients. ETBF represents a control bft positive strain from ATCC. (A–D), *p < 0.05. ***p < 0.001. One-way ANOVA with multiple comparisons. (E) Ratio of IL10/IL-12p40 expression detected from co-culturing B. fragilis supernatants with monocytic cell line by ELISA. (F) Ratio of IL10/IL-12p40 expression in biopsy tissue samples detected by ELISA. See also Figure S4.

Figure 4.. Persistent NTBF isolates from patients with polyps are proteomically distinct and enriched with LPS biosynthesis genes

(A) Fold change NF-kB stimulation of HEK293 cells transfected with TLR2 and infected with supernatants from B. fragilis isolates relative to unstimulated control in relative light unit (RLU). Results are means ± SEM from 3 independent experiments including 16 PA and 16 NPA isolates from 4 different TAP patients, 8 PA and 8 NPA clinical isolates from 2 different SSP patients, and 4 clinical isolates from 1-PF patient. ETBF+ represents the bft positive study isolates that include 4 PA and 4 NPA isolates from 1 TAP patient and 8 isolates from 2 PF patients (by one-way ANOVA with multiple comparisons, *p < 0.05). (B) Fold NF-kB stimulation of HEK293 cells transfected with TLR4 and infected with B. fragilis isolates supernatants relative to unstimulated control. Results are means ± SEM from 3 independent experiments (by one-way ANOVA with multiple comparisons, *p < 0.05). (C) Principal component analysis based on peptide mass fingerprint profile of various NTBF B. fragilis isolates. Data are mean ± SEM of ± of 2–3 independent experiments with n = 4–6. Each dot represents the average of technical replicates. Differences are maintained after growing isolates multiple times. (D) Prevalence of clusters of orthologous proteins based on comparative genome analysis between isolates from patients with polyps and polyp-free patients. (E) LPS expression revealed by immunohistochemistry (IHC) in CRC patients’ biopsies (40×). Comparison of PF human normal colon tissue section and NTBF positive SSP and TAP tumor sections. Representative picture for 3–4 sections from one biopsy from each type of tissue (PF, SSP, or TAP), no quantitative data available due to tissue restriction. (F) Percentage of glycosyltransferase gene present in the host gut tissue based on PCR performed on 7 patients with TAP, 6 patients with SSP, and 9-PF patients. See also Figure S4.