A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche

Abstract

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals¹, is enriched in human colorectal cancer (CRC) tumours^2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis^5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.

Fig. 1. Fn niche features.

a, A schematic of Fusobacterium strain collection (n = 146) and the sequencing strategy for unique strains. SMRT, single-molecule real-time sequencing. b, A column graph depicting the proportion of Fusobacterium genomes, subset by species, within the CRC (orange) and oral (blue) niches. The inset shows all non-Fn species of Fusobacterium (Fnec, F. necrophorum; Fu, F. ulcerans; Fp, F. pseudoperiodonticum; Fc, F. canifelinum; Fv, F. varium). c, The composition of the Fn pangenome subset by niche. Anvi’o gene cluster (GC) prevalence was used to define core (≥95%), accessory (≥5% and <95%) and rare (<5%) features conserved in both CRC-associated and oral-associated strains (collection core, ≥95% in all strains within the collection; collection cloud, ≥5% and <95% in all strains within the collection; collection rare, <5% in all strains within the collection). Disparate features are those that do not fall into any of the other noted bins. d, The proportion of niche-enriched gene clusters across CRC-associated and oral-associated Fn genomes. The plot box shows the 25th percentile, median and 75th percentile. The plot whiskers indicate the minima and maxima. e, KofamKOALA KEGG orthologue analysis of niche-enriched gene clusters. f, A column graph depicting the proportion of Fn genomes, grouped by subspecies, within the CRC and oral niche. Statistical analysis was carried out using a two-sample z-test, two-tailed. NS, not significant. g, Gene presence–absence heat map of canonical Fn virulence factors (fadA (refs. ^,,), fap2 (ref. ), fplA (ref. ), radD (refs. ^,), aim1 (ref. ), cmpA (ref. ) and fusolisin) across Fn subspecies, in which each column represents an individual genome (Fna n = 75, Fnn n = 17, Fnp n = 33, Fnv n = 10). The heat map is organized using an rpoB gene-based phylogenetic tree. For each genome, the tree end points indicate the niche origin (CRC (orange); oral (blue)) and the bar colour indicates the Fn subspecies (Fna (red); Fnn (gold); Fnp (purple); Fnv (brown)). The graphics in a were created using BioRender.com.

Fig. 2. Genetic and epigenetic characteristics of Fna clades.

a, A kSNP maximum-likelihood whole-genome phylogenetic tree. For each Fn genome (n = 135), the tree end points indicate the niche origin (CRC (orange); oral (blue)) and the bar colour indicates the Fn subspecies (Fna (red); Fnn (gold); Fnp (purple); Fnv (brown)). Within Fna, the background colour indicates the Fna clade (Fna C1 (green); Fna C2 (lavender)). b, A clustered dendrogram of the ANI matrix. The bar colour indicates the Fn subspecies (Fna (red); Fnn (gold); Fnp (purple); Fnv (brown)). The Fna clades are highlighted with green and lavender boxes. ANI values are reported in Supplementary Tables 4 and 5. c, A GiG-map visualization of the protein-coding gene content across Fn genomes. The top bar colour indicates the Fn subspecies (Fna (red); Fnn (gold); Fnp (purple); Fnv (brown)) and the box colour indicates the Fna clade (Fna C1 (green); Fna C2 (lavender)). The inset on the right highlights groups of protein-coding genes that are distinct between Fna C1 and Fna C2. An interactive GiG-map dataset is available at https://fredhutch.github.io/fusopangea/. d, PCA of Anvi’o gene clusters by presence and absence in each genome. The colours indicate the Fn subspecies and Fna clades (Fnn (gold); Fnp (purple); Fnv (brown); Fna C1 (green); Fna C2 (lavender)). The ellipses are drawn to 95% confidence. e, Left: PCA of Fna genome-wide methyl-modified nucleotide sequences. The ellipses are drawn to 95% confidence. The overlay of the PCA biplot shows the top five nucleotide motifs that are driving the Fna clade bifurcation. Right: a table indicating the distribution of each motif across the Fna clades. The colour indicates the Fna clade (Fna C1 (green); Fna C2 (lavender)). f, A column graph depicting the proportion of Fna CRC-associated and Fna oral-associated genomes, subset by Fna clade (Fna C1 (green); Fna C2 (lavender)). The statistical analysis was carried out using a two-sample z-test, two-tailed. NS, not significant. The graphics in f were created using BioRender.com.

Fig. 3. Inter Fna-clade comparative analyses.

a, A gene presence versus absence column graph depicting the proportion of Fna genomes containing canonical Fn virulence factors, subset by clade (Fna C1 (green); Fna C2 (lavender)). The statistical analysis was carried out using a two-sample z-test, two-tailed. b, Left: computational confocal analysis of colon cancer epithelial cells (HCT116; grey) co-incubated with representative Fna C1 (green) or Fna C2 (lavender) strains. Scale bars, 4 μm. Right: a bar plot demonstrating the percentage of HCT116 cells with intracellular Fna; n = 3 biological replicates with 3 analysed z-stacks. Data are plotted as mean ± s.e.m. The statistical analysis was carried out using Welch’s t-test, two-tailed. c, A PPanGGOLiN map of the Fna pangenome. Each node represents a gene group, syntenic nodes represent neighbouring genes, the size indicates relative presence across Fna genomes, and the colour depicts pangenome partition (Fna core (red); Fna C1 accessory genome (green); Fna C2 accessory genome (lavender)). The white arrows (left panel) and oblongs (right panel) indicate Fna C2-associated putative eut and pdu operons. d, Schematics of these Fna C2 operons. An interactive PPanGGOLiN map is available at https://fredhutch.github.io/fusopangea/. e,f, Differentially expressed genes (log₂[fold change] ≥ 0.58 and ≤−0.58 with −log₁₀[P value] ≥ 1.30) in a representative Fna C2 strain, SB010, exposed to EA (e) or 1,2-PD (f) compared to unexposed SB010 control. To highlight SB010-unique content, genes also differentially expressed under the same exposure conditions in a representative Fna C1 strain, KCOM 3764, have been removed (Extended Data Fig. 4b,c). The vertical dotted lines indicate the threshold of significant gene expression, defined as log₂[fold change] ≥ 0.58 and ≤−0.58. The statistical analysis was carried out using glmQLFTest, two-sided. The data point colours indicate Fna core (red), Fna C1 accessory (green), Fna C2 accessory (lavender) or Fna cloud (black; present in ≥5% and <95% in all Fna strains) genes. The stars indicate eut and pdu operon genes.

Fig. 4. Fna C2 impact on intestinal tumorigenesis and metabolism.

a, A schematic of the study with 6–8-week-old Apc^Min+/− mice receiving streptomycin and dextran sodium sulfate (DSS) treatment to alter the native microbiome and induce colitis, respectively. Mice were orally gavaged with vehicle control (arm 1) or a mix of three representative Fna C1 (arm 2) and Fna C2 (arm 3) strains. A strain mix was used to capture a higher proportion of Fna clade-specific accessory genes (Extended Data Fig. 2d). The mice were monitored until the end-point at 6 weeks post-gavage when they reached 15–17 weeks of age. b, A plot indicating the number of adenomas in the large intestine by treatment arm (vehicle control (grey); Fna C1 treated (green); Fna C2 treated (lavender); n = 8 mice per arm). The data are plotted as mean ± s.e.m. The statistical analysis was carried out using one-way ANOVA. c, Partial least squares discriminant analysis of detected intestinal metabolites (n = 1,296). The colours represent the treatment arm (vehicle control (grey); Fna C1 treated (green); Fna C2 treated (lavender)). The graphics in a were created using BioRender.com. Source Data

Fig. 5. Fn in human tissue microbiome and stool metagenomic specimens.

a, Plots showing the relative abundance for Fusobacterium species (Fg, F. gonidiaformans; Fh, F. hwasookii; Fm, F. mortiferum; Fnavi, F. naviforme; left plot), and Fn subspecies and Fna clades (right plot) using microbial 16S rRNA gene sequencing of paired tumour (orange) and normal adjacent (black) tissue (n = 62 patients with CRC). Amplicon sequence variants were used to obtain Fna clade resolution (Extended Data Fig. 10 and Supplementary Table 8). The data are plotted as mean ± s.e.m. The statistical analysis was carried out using one-sided t-test, paired. b, Plots showing the relative abundance for Fna C1 (green) and Fna C2 (lavender) within patient primary colorectal tumour tissue from two independent cohorts (cohort 1 (n = 116) this study; cohort 2 (n = 86) BioProject PRJNA362951). The data are plotted as mean ± s.e.m. The statistical analysis was carried out using one-sided t-test, paired. c, Fna C1 and Fna C2 detection in stool metagenomic data from patients with CRC and healthy individuals. The left plot shows the pooled effect sizes for Fna C1 (green) and Fna C2 (lavender) calculated using a meta-analysis of standardized mean differences and a random-effects model on MetaPhlAn4 (ref. ) species-level genome bin abundances on all CRC samples (n = 627) and samples from healthy individual (n = 619). The right plot shows the effect sizes for Fna C1 and Fna C2 calculated using the same approach, but here samples in which Fna C1 co-occurred with Fna C2 were excluded. The data are plotted as mean ± s.e.m. The statistical significance was assessed by a Wald test, two-sided. All P values are corrected using the Benjamini–Yakuteli method. d, Fna C1 and Fna C2 presence in stool metagenomes of patients with CRC. The bars indicate individual stool samples from patients with CRC (n = 627) and are coloured by Fna C1 and Fna C2 detection (Fna C1 detected (green); Fna C2 detected (lavender); Fna undetected (grey)). The lower brackets indicate the number of stool samples in which Fna C1 occurred independently (n = 5), Fna C2 occurred independently (n = 147), Fna clades co-occurred (n = 31) or Fna clades were not detected (n = 444). The graphics in a–c were created using BioRender.com.

Extended Data Fig. 1. Fn genetic characterization by niche and subspecies.

a, Size of the Fn pangenome split by the core genome (≥95%) (black) and accessory (<95%) genome (grey). n = 10,000 random subsamplings of 135 Fn genomes. Data is plotted as median ± s.d. b, Size of the Fn pangenome split by CRC-associated (orange) and oral-associated (blue) niche origin, with respective core and accessory genomes labeled. n = 10,000 random subsamplings of 55 Fn CRC-associated and 80 Fn oral-associated genomes. Data is plotted as median ± s.d. c, Size of the Fn pangenome split by Fn subspecies, Fna (red), Fnn (gold), Fnp (purple), Fnv (brown), with respective core and accessory genomes labeled. n = 10,000 random subsamplings of 75 Fna, 17 Fnn, 33 Fnp, and 10 Fnv genomes. Data is plotted as median ± s.d. d-e, Maximum-likelihood dendrograms of d, fadA and e, fplA nucleotide and amino acid sequences. For each genome, tree end points indicate Fn subspecies; Fna (red), Fnn (gold), Fnp (purple), Fnv (brown). f, Column graph depicts the proportion of Fn genomes containing canonical Fn virulence factors, subset by Fna (red), non-Fna (black) subspecies. Statistical analysis performed using two sample Z test, two-tailed. NS, not significant.

Extended Data Fig. 2. Phylogenetics and genomics of Fna clades.

a, Maximum-likelihood phylogenetic trees of Fn single marker genes, 16 S rRNA, rpoB, zinc protease, nusA and nusG. For each genome (n = 135), tree end points indicate niche origin, CRC (orange) or oral (blue), bar color indicates Fn subspecies (Fna (red), Fnn (gold), Fnp (purple), Fnv (brown)), and background color indicates Fna clades (Fna C1 (green) and Fna C2 (lavender)). b, Genes-in-genomes map (GiG-map) visualization of protein coding gene content across Fna genomes. Boxes highlight Fna clades, Fna C1 (green) and Fna C2 (lavender). Previously published NCBI genomes are labeled by strain name. c, Size of the Fna pangenome split by Fna clade, Fna C1 (green) and Fna C2 (lavender), with respective core and accessory genomes labeled. n = 10,000 random subsamplings of 24 Fna C1 and 51 Fna C2 genomes. Data is plotted as median ± s.d. d, Composition of Fna pangenome subset by clade. Anvi’o gene cluster (GC) prevalence was used to define core (≥95%), accessory (≥5% and <95%), and rare (<5%) features conserved in both Fna C1 and Fna C2 strains (“Fna core” (≥95% in all Fna strains), “Fna cloud” ( ≥ 5% and <95% in all Fna strains), “Fna rare” (<5% in all strains) or unique for strains from each clade. Disparate features are those that do not fall into any of the other noted bins. Plot box shows 25^th percentile, median, and 75^th percentile. Plot whiskers indicate minima and maxima. e, Column graph indicates chromosome sizes in Fna C1 (n = 24) and Fna C2 (n = 51). Data is plotted as mean ± s.e.m. Statistical analysis performed using Welch’s T-test, two-tailed. f, Column graph depicts the proportion of Fna genomes containing innate bacterial genetic defense systems, subset by Fna clades, Fna C1 (green) and Fna C2 (lavender). Statistical analysis performed using two sample Z test, two-tailed. NS, not significant. g, Graph shows the percent relative abundance of Fna C1 (green) and Fna C2 (lavender) in paired saliva (circle) or tumor biopsy (triangle) samples from 39 patients with colorectal adenocarcinomas. Data is plotted as mean ± s.e.m. Statistical analysis performed using Welch’s T-test, paired. NS, not significant.

Extended Data Fig. 3. Morphological and genomic differences between Fna clades.

Representative Fna C1 and Fna C2 strains co-cultured with human colon cancer cells (HCT116). a-b, Computational analysis of confocal imaging. Independent masks for cancer epithelial cells (grey), and intracellular bacterial cells (Fna C1 green; Fna C2 lavender) were generated. Masks were used to calculate the percent of HCT116 cells with intracellular Fna (Fig. 3b) (see Methods). Scale bar is 20 μm. c, Bacterial aerotolerance was assessed through serial dilution plating at start, mid-point, and endpoint of co-culture. Graph shows resulting bacterial colony forming units per millilter, standardized to start point for each strain. Dashed line indicates normalization equal to one. Statistical analysis performed using a Welch’s T-test, two-tailed. d, Bar plots indicate Fna cell length and cell width as measured from confocal microscopy images, subset by Fna clades, Fna C1 (green) or Fna C2 (lavender); n = 45 cells per Fna clade. Data is plotted as mean ± s.e.m. Statistical analysis performed using Welch’s T-test, two-tailed. e, KofamKOALA KEGG ortholog mapping of Fna clade-enriched gene clusters.

Extended Data Fig. 4. Fna clade transcriptomic responses to intestinal metabolites.

a, Bar plots demonstrate the proportion of stool metagenomic samples from patients with CRC or healthy controls in which putative eut and pdu operons were detected. Statistical analysis performed using two sample Z test, two-tailed. Cohort sample sizes are indicated at the bottom of each panel. b-c, Differentially expressed genes (with log2-transformed fold change ≥ 0.58 and ≤ −0.58 with -log10(p-value) ≥ 1.30) of a representative Fna C1 strain (KCOM 3764) and a representative Fna C2 strain (SB010) under (b) ethanolamine (EA) or (c) 1,2-propanediol (1,2-PD) exposure as compared to their respective unexposed controls. Top five significant (-log10(p-value) ≥ 1.30) upregulated and downregulated genes are labeled. d, Differentally expressed genes (with log2-transformed fold change ≥ 0.58 and ≤ −0.58 with -log10(p-value) ≥ 1.30) in SB010 under Vitamin B12 exposure alone as compared to unexposed control. All differentially expressed genes labeled. For b-d, vertical dotted lines indicate the threshold of significant gene expression, defined as log2-transformed fold change ≥ 0.58 and ≤ −0.58. Statistical analysis performed using glmQLFTest, 2-sided. Data point colors indicate whether a gene is categorized as part of the Fna core genome (red), Fna C1-associated accessory genome (green), or Fna C2-associated accessory genome (lavender) by PPanGGOLiN.

Extended Data Fig. 5. Differences in pH preference and acid resistance across Fna clades.

a, Schematic of potential gastrointestinal route from the oral primary niche to the CRC tumor secondary niche. b, Plot indicates growth activity as measured in Biolog PM10 plates for representative Fna C1 (green) and Fna C2 (lavender) strains. Data is plotted as the normalized average across duplicates. Statistical analysis at each pH performed using Welch’s T-test, two-tailed. NG = no growth. c, PPanGGOLiN map of Fna pangenome. Each node represents a gene group, syntenic nodes represent neighboring genes, size indicates relative presence across Fna genomes, and color depicts elements in the Fna C1-associated accessory genome (green), and the Fna C2-associated accessory genome (lavender). White arrow indicates putative glutamate-dependent acid resistance (gdar) operon. d, Schematic depicts mechanism of GDAR acid resistance system. e, Qualitative and f, quantitative measurements of colorimetric assay measuring pH change indicative of conversion of glutamine to glutamate (yellow to green) and conversion of glutamate to γ-aminobutyric acid (GABA) (green to blue) in the presence of representative Fna C1 and Fna C2 strains. n = 3 technical triplicates of 3 biological replicates of 3 strains per Fna clade. Data is plotted as mean ± s.e.m and statistical analysis performed using an ANOVA. g, Schematic of experiment testing the effects of pH stress by exposure to simulated gastric fluid (SGF) at pH 3 or SGF supplemented with 10 mM glutamate at pH 3. Plates show resulting growth for a representative Fna C1 and a representative Fna C2 strain over the course of an hour exposure, as compared to tryptic soy broth (TSB) control at pH ~6.7. h, Bar plots demonstrate the proportion of stool metagenomic samples from patients with CRC (orange) or healthy controls (black) in which a putative gdar operon was detected. Statistical analysis performed using two sample Z test, two-tailed. Cohort sample sizes are indicated at the bottom of each panel. The graphics in a,d,g were created using BioRender.com.

Extended Data Fig. 6. Intestinal adenoma burden and fecal Fusobacterium load in Fna treated mice.

a, Schematic of study with Apc^Min+/− mice orally gavaged with vehicle control (Arm 1) or representative Fna C1 (Arm 2) and Fna C2 (Arm 3) strains post streptomycin and dextran sodium sulfate (DSS) to clear the native microbiome and induce colitis, respectively. b-c, Plots indicate the number of adenomas in b, the small and large intestines combined and c, the small intestine by treatment arm, vehicle control (grey), Fna C1-treated (green), or Fna C2-treated (lavender). Data is plotted as mean ± s.e.m; n = 8 mice per treatment arm. Statistical analysis performed using one-way ANOVA. d, Fusobacterium-targeted qPCR was performed on fecal pellets from one-day post-gavage (PG) to study endpoint. Fn presence in each sample is plotted as Fn copies per ng of fecal DNA with a detection limit (D.L.) of 1 Fn copy. Each sample was run in triplicate, and samples with ≥2 duplicates with detectable signal are included. The graphics in a were created using BioRender.com. Source Data

Extended Data Fig. 7. Intestinal metabolite changes in Fna treated mice.

Scatter plots shows fold change for 1,296 detected metabolites from a, Fna C2 treated mice compared to control, b, Fna C2 treated mice compared to Fna C1 treated mice, and c, Fna C1 treated mice compared to control colored by metabolic categorization. Top five characterized metabolites are labeled. Plots below indicate the pathway enrichment score all enriched pathways. Bars are colored by metabolic categorization.

Extended Data Fig. 8. Altered metabolites in Fna treated mice.

a, Plots show the log2-transformed fold change in metabolite level between treatment arms by the -log10(p-value). Points indicate individual metabolites colored by their metabolic categorization. Dotted lines indicate the threshold of significant gene expression, defined as log2-transformed fold change ≥ 0.58 and ≤ −0.58 (vertical lines) and a -log10(p-value) ≥ 1.30 (horizontal line). Statistical analysis performed on natural log-transformed values using one-sided T-test. b, Clustered heatmap of top fifty metabolites across study arms. Dendrogram groups individual samples by similarity of metabolite profile. Bar color for each sample indicates treatment arm, vehicle control (grey), Fna C1-treated (green), or Fna C2-treated (lavender). c, Schematic of glutathione metabolic pathway. d, Plot demonstrates the ratio between oxidized (GSSG) and reduced (GSH) glutathione levels for each treatment arm, vehicle control (grey), Fna C1-treated (green), or Fna C2-treated (lavender); n = 4 mice per arm. Data is plotted as mean ± s.e.m. Statistical analysis performed using one-way ANOVA. e, Schematic of eicosanoid metabolic pathway.

Extended Data Fig. 9. Significantly altered intestinal metabolites in Fna C2-treated mice.

Plots of individual, characterized metabolites, with metabolic categories having > 10 significantly altered metabolites shown. Each plot shows the log2-transformed log change between Fna C2-treated mice compared to Fna C1-treated mice versus the log2-transformed log change between Fna C2-treated mice compared to control mice. Dashed lines indicate the threshold of significantly altered metabolites, defined as log2-transformed fold change (FC) ≥ 0.58 and ≤ −0.58. Upper right quadrant indicates metabolites that are significantly elevated in Fna C2-treated mice as compared to both Fna C1-treated and control mice. Lower left quadrant indicates metabolites that are significantly lower in Fna C2-treated mice as compared to both Fna C1-treated and control mice. Top metabolites in each of these quadrants are labeled.

Extended Data Fig. 10. Detection of Fn subspecies and Fna clades in human stool metagenomes.

Detection of a, Fn subspecies (Fnn, Fnv, Fnp) and a-k, Fna clades (Fna C1 and Fna C2) in stool metagenomic data from previously published independent cohorts from patients with CRC and healthy controls. a, Plot shows the percent relative abundance of each Fn subspecies and Fna clade in stool samples from patients with CRC (n = 627) or healthy controls (n = 619). Data is plotted as mean ± s.e.m and statistical analysis performed using a one-way ANOVA. b-k, Samples are plotted both by (b-k) individual cohorts and (k) pooled results. In each panel, top plot shows the percent relative abundances of Fna C1 (green) and Fna C2 (lavender) in each sample. Data is plotted as mean ± s.e.m and statistical analysis performed using Welch’s T-test, paired. Bottom plot demonstrates the proportion of stool samples from patients with CRC and the proportion of stool samples from healthy controls in which Fna C1 and Fna C2 were detected. Data is plotted as mean + s.d. and statistical analysis performed using two sample Z test, two-tailed. Cohort sample sizes are indicated at the bottom of each panel.

Extended Data Fig. 11. Meta-analysis of Fna in human stool metagenomes.

Fna C1 and Fna C2 detection in stool metagenomic data from previously published independent cohorts from patients with CRC and healthy controls plotted by individual cohorts. Left plot shows the effect sizes for Fna C1 and Fna C2 calculated across all samples (CRC n = 627, healthy control n = 619) using a meta-analysis of standardized mean differences and a random effects model on MetaPhlAn4 species-level genome bins (SGB) abundances. Right plot shows the effect sizes for Fna C1 and Fna C2 calculated using the same approach but with samples where Fna C1 co-occurred with Fna C2 excluded (CRC n = 596, healthy control n = 616). Data is plotted as mean ± s.e.m. Statistical significance assessed by Wald test, two-sided. All p-values are corrected via the Benjamini-Yakuteli method.