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) tumours2–5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5–8. 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.

Tangled genetic relationships within theFusobacteriumgenus

“…Nonetheless, all analyses confirmed that four genomes that include strains FNU and 13-08-02 (in clade C1 in Zepeda-Rivera et al) do not belong to the F. nucleatum animalis subspecies or any other known species/subspecies. Thus, our data do not support the previously suggested division of F. nucleatum animalis diversity in two clades 6 . In their recent study, Zepeda-Rivera and coworkers showed that clade C2 was associated to the CRC niche, whereas C1 was not.…”

“…Indeed, all these features were compared between two distinct species, although closely related. Whereas this is unlikely to change the conclusion that F. nucleatum animalis is associated with CRC, our data call for a re-assessment of the characteristics of clade C2, which were established in comparison to a different species 6 . This is particularly true in light of the PCA and STRUCTURE analyses, which indicated that the new specie/clade C1 and F. nucleatum animalis belong to different clusters or modern populations.…”

“…A different lineage (represented by F. varium and F. ulcerans) was instead associated with lymphovascular invasion 9 . Conversely, another study found enrichment of F. varium in CRC samples 8 , whereas F. nucleatum animalis or even a specific clade within the diversity of this subspecies was found to be overabundant in the CRC niche in different studies 5,6,42 . Finally, F. necrophorum and F. nucleatum were found to colonize both primary CR tumors and the corresponding liver metastases 3 .…”

“…Fusobacterium species have gained enormous interest in relation to their oncogenic role in colorectal cancer (CRC) and other tumor types. In particular, most studies have focused on Fusobacterium nucleatum, which was shown to be enriched in the gut microbiota of CRC patients and to promote carcinogenesis through multiple mechanisms [1][2][3][4][5][6] . F. nucleatum (and F. necrophorum) was also found in the lymph node and liver metastases of Fusobacterium-associated primary tumors 3 .…”

“…These subspecies are phylogenetically divergent to the point that they were suggested to represent distinct species 11,12 . Also, a recent report suggested that F. nucleatum subspecies animalis is divided into two clades, only one of which is associated with CRC 6 . Finally, several works identified species miss-classifications in public records, whereas some Fusobacterium genomes cannot be assigned to any existing species 9,13 .…”