A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche
Martha Zepeda-Rivera et al.
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) tumours2,3,4,5. High intratumoral Fn loads are associated with recurrence, metastases and poorer patient prognosis5,6,7,8. 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.
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. 63) 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.
